WD Repeat Research Articles

Receptor for Activated C Kinase 1 counteracts ABSCISIC ACID INSENSITIVE5-mediated inhibition of seed germination and post-germinative growth in Arabidopsis.

ABSCISIC ACID INSENSITIVE5 (ABI5), a key regulator of the abscisic acid (ABA) signalling pathway, plays a fundamental role in seed germination and post-germinative development. However, the molecular mechanism underlying the repression function of ABI5 remains to be elucidated. In this study, we demonstrate that the conserved eukaryotic WD40 repeat protein Receptor for Activated C Kinase 1 (RACK1) is a novel negative regulator of ABI5 in Arabidopsis. The RACK1 loss-of-function mutant is hypersensitive to ABA, while this phenotype is rescued by a mutation in ABI5. Moreover, overexpression of RACK1 suppresses ABI5 transcriptional activation activity for ABI5-targeted genes. RACK1 may also physically interact with ABI5 and facilitate its degradation. Furthermore, we found that RACK1 and the two substrate receptors CUL4-based E3 ligases (DWA1 and DWA2) function together to mediate the turnover of ABI5, thereby efficiently reducing ABA signalling in seed germination and post-germinative growth. In addition, molecular analyses demonstrated that ABI5 may bind to the promoter of RACK1 to repress its expression. Collectively, our findings suggest that RACK1 and ABI5 might form a feedback loop to regulate the homeostasis of ABA signalling in acute seed germination and early plant development.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

A nuclear WD40 repeat protein PRL1 regulates stability of MYB4 transcription factor in Arabidopsis

MYB4, a member of the R2R3-type subfamily of MYB transcription factor plays a crucial role in regulating the accumulation of UV-B absorbing phenylpropanoids in plants. UV-B exposure for a longer duration down-regulates the expression of MYB4 gene in Arabidopsis. MYB4 protein represses its own expression by binding to its own promoter. However, at present practically nothing is known about the post-translational regulation of MYB4 protein in vivo. Here, we provide evidence that in Arabidopsis MYB4 protein is phosphorylated in vivo and is targeted by the ubiquitin-26S proteasome-dependent pathway. Immunoprecipitation, immunoblotting, and phosphoprotein staining experiments have revealed that both the accumulation pattern and phosphorylation of MYB4 increase in the light condition during the 24 hours time span under long-day conditions. Yeast two-hybrid and bimolecular fluorescence complementation assays have shown that MYB4 directly interacts with a nuclear WD40 repeat protein, PRL1 in vivo. Cell-free protein degradation assay in the absence and presence of proteasome inhibitor indicates that MYB4 is degraded in a ubiquitin proteasome-dependent manner. Furthermore, analyses of MYB4 protein accumulation levels in transgenic atmyb4–1 mutant line expressing 35 S:AtMYB4 (35 S:AtMYB4-atmyb4–1) and atprl1–1 mutant line indicate that PRL1 regulate stability of MYB4 in Arabidopsis. Overall, our results provide important information on the possible mechanism of post-translational modification and regulation of stability of MYB4 protein in Arabidopsis in vivo.

Phylogeny and Expansion of Serine/Threonine Kinases in Phagocytotic Bacteria in the Phylum Planctomycetota.

The recently isolated bacterium "Candidatus Uabimicrobium amorphum" is the only known prokaryote that can engulf other bacterial cells. Its proteome contains a high fraction of proteins involved in signal transduction systems, which is a feature normally associated with multicellularity in eukaryotes. Here, we present a protein-based phylogeny which shows that "Ca. Uabimicrobium amorphum" represents an early diverging lineage that clusters with the Saltatorellus clade within the phylum Planctomycetota. A gene flux analysis indicated a gain of 126 protein families for signal transduction functions in "Ca. Uabimicrobium amorphum", of which 66 families contained eukaryotic-like Serine/Threonine kinases with Pkinase domains. In total, we predicted 525 functional Serine/Threonine kinases in "Ca. Uabimicrobium amorphum", which represent 8% of the proteome and is the highest fraction of Serine/Threonine kinases in a bacterial proteome. The majority of Serine/Threonine kinases in this species are membrane proteins and 30% contain long, tandem arrays of WD40 or TPR domains. The pKinase domain was predicted to be located in the cytoplasm, while the WD40 and TPR domains were predicted to be located in the periplasm. Such domain combinations were also identified in the Serine/Threonine kinases of other species in the Planctomycetota, although in much lower abundances. A phylogenetic analysis of the Serine/Threonine kinases in the Planctomycetota inferred from the Pkinase domain alone provided support for lineage-specific expansions of the Serine/Threonine kinases in "Ca. Uabimicrobium amorphum". The results imply that expansions of eukaryotic-like signal transduction systems are not restricted to multicellular organisms, but have occurred in parallel in prokaryotes with predatory lifestyles and phagocytotic-like behaviors.

SCFFBXW11 Complex Targets Interleukin-17 Receptor A for Ubiquitin-Proteasome-Mediated Degradation.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that participates in innate and adaptive immune responses and plays an important role in host defense, autoimmune diseases, tissue regeneration, metabolic regulation, and tumor progression. Post-translational modifications (PTMs) are crucial for protein function, stability, cellular localization, cellular transduction, and cell death. However, PTMs of IL-17 receptor A (IL-17RA) have not been investigated. Here, we show that human IL-17RA was targeted by F-box and WD repeat domain-containing 11 (FBXW11) for ubiquitination, followed by proteasome-mediated degradation. We used bioinformatics tools and biochemical techniques to determine that FBXW11 ubiquitinated IL-17RA through a lysine 27-linked polyubiquitin chain, targeting IL-17RA for proteasomal degradation. Domain 665-804 of IL-17RA was critical for interaction with FBXW11 and subsequent ubiquitination. Our study demonstrates that FBXW11 regulates IL-17 signaling pathways at the IL-17RA level.

Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

Abstract 2640: HBI-2375, a first-in-class IND ready selective MLL1-WDR5 inhibitor, retains desirable preclinical characteristics in solid tumors and in leukemias to be pursued in clinical studies

Abstract MLL1 (mixed lineage leukemia1) interaction with WDR5 (WD repeat domain 5) is critical for its histone methyl transferase (HMT) activity leading to transcriptional regulation of gene expression. MLL1 dysregulation such as its translocation at chromosome 11q23 results in generation of MLL1 fusion genes and leukemia development. Additionally, recent studies indicated a role for MLL1-WDR5 interaction in solid tumors progression via directly affecting cancer cells or by suppression of tumor immunity. Therefore, MLL1-WDR5 interaction is an important target in the discovery of anti-cancer drugs for both leukemia and solid tumors. We recently identified HBI-2375 as a selective inhibitor of MLL1-WDR5 interaction. HBI-2375 showed potent inhibition of WDR5 (IC50: 4.48nM) in biochemical assay and inhibited MV4-11 cellular proliferation (average IC50: 3.17µM) in CTG assays. In addition, HBI-2375 was stable in liver S9 and whole blood, with moderate to high plasma protein binding rates in multiple species, and acceptable hERG (IC50: 17µM). Furthermore, HBI-2375 displayed reasonable oral PK properties (Cmax, T1/2, AUC, and F%) in mouse, rat and dog and showed good safety profile in GLP tox studies. In pharmacology studies HBI-2375 showed significant dose dependent anti-tumor activity in MV4-11 AML model along with reduction in methylation of H3K4 in the tumors harvested from the treated animals demonstrating the targeted disruption of HMT activity in cancer cells by HBI-2375. Furthermore HBI-2375 in combination with PD-1 mAb significantly inhibited tumor growth in MC38, colorectal tumor model, and in 3LL lung carcinoma syngeneic tumor models. Flow cytometry and IHC showed significant increase in the infiltration of CD8+ CTLs in the combination group further providing immune-driven mechanisms for greater efficacy of combination therapy. Gene expression analysis also identified genes that are involved in promoting immune system activity in the combination group. Tissue distribution studies also showed greater accumulation of HBI-2375 in brain tissue indicative of a potential to target glioma and glioblastoma in clinical studies. Therefore, HBI-2375 represents a first-in-class MLL1-WDR5 inhibitor with promising characteristics for future testing in the clinic. Citation Format: Farbod Shojaei, Che Fang, Jill M. Ricono, Mireille Gillings. HBI-2375, a first-in-class IND ready selective MLL1-WDR5 inhibitor, retains desirable preclinical characteristics in solid tumors and in leukemias to be pursued in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2640.

The clinical relevance of WDFY4 in autoimmune diseases in diverse ancestral populations.

WD repeat- and FYVE domain-containing protein 4 (WDFY4), coded by a gene on 10q11.23, is a member of the BEACH (Beige and Chediak-Higashi) domain-containing family. Genome-wide association studies identified WDFY4 variants as a risk factor for SLE in Asian and European populations. WDFY4 variants are also associated with RA and primary biliary cholangitis, in different ancestry populations. The WDFY4 protein plays an essential role in the cross-presentation of classic dendritic cells, reactive oxygen species-induced apoptosis of CD8+ T cells, and non-canonical autophagic activity in B cells. A novel variant rs7919656 was identified in Japanese clinically amyopathic dermatomyositis patients, with a highly expressed truncated isoform augmenting the melanoma differentiation-associated gene 5 (MDA5) signalling pathway. The same variant was later found to be significantly associated with RP-ILD in Chinese MDA5+DM patients. Here, we briefly review the association of WDFY4 with autoimmune diseases and its known function in immune response.

WDR4 promotes HCC pathogenesis through N7-methylguanosine by regulating and interacting with METTL1

BackgroundThe N7-methylguanosine (m7G), a modification at defined internal positions within tRNAs and rRNAs, is correlated with tumor progression. Methyltransferase like 1 (METTL1)/ WD repeat domain 4 (WDR4) mediated tRNA m7G modification, which could alter many oncogenic mRNAs translation to promote progress of multiple cancer types. However, whether and how the internal mRNA m7G modification is involved in tumorigenesis remains unclear. MethodsThe immunohistochemistry assay was conducted to detect the expression of WDR4 and METTL1 in hepatocellular carcinoma (HCC) and the expression of both genes whether contributes to the prognosis of the survival rate of HCC patients. Then, CCK8, colony formation assays and tumor xenograft models were conducted to determine the effects of WDR4 on HCC cells in vitro and vivo. Besides, dot blot assay, m7G-MeRIP-seq and RNA-seq analysis were conducted to determine whether WDR4 contributes to m7G modification and underlying mechanism in HCC cells. Finally, rescue and CO-IP assay were conducted to explore whether WDR4 and METTL1 proteins form a complex in Huh7 cells. ResultsWDR4 modulates m7G modification at the internal sites of tumor-promoting mRNAs by forming the WDR4-METTL1 complex. WDR4 knockdown downregulated the expression of mRNA and protein levels of METTL1 gene and thus further modulate the formation of WDR4-METTL1 complex indirectly. METTL1 expression was markedly correlated with WDR4 expression in HCC tissues. HCC patients with high expression of both genes had a poor prognosis. ConclusionsWDR4 may contribute to HCC pathogenesis by interacting with and regulating the expression of METTL1 to synergistically modulate the m7G modification of target mRNAs in tumor cells.

Molecular Cloning, Characterization, and Expression of a Receptor for Activated Protein Kinase C1 (RACK1) Gene in Exopalaemon carinicauda Zoea Larvae under Aroclor 1254 Stress.

The receptor for activated protein kinase C1 (RACK1) belongs to the typical WD repeat family, which is extremely conservative and important in multiple signal transduction pathways related to growth and development that coordinate the intracellular role of various life activities. As a novel protein with versatile functions, it was found in a variety of organisms. In a previous study, we identified the RACK1 sequence of white shrimp from transcriptome data. In this study, we employed specialized bioinformatics software to conduct an in-depth analysis of EcRACK1 and compare its amino acid sequence homology with other crustaceans. Furthermore, we investigated the expression patterns of RACK1 at different developmental stages and tissues, as well as at various time points after exposure to Aroclor 1245, aiming to elucidate its function and potential response towards Aroclor 1245 exposure. The length of EcRACK1 is 957 nucleotides, which encodes 318 amino acids. Moreover, there were seven typical WD repeats in EcRACK1, which have more than a 96% sequence identity with the RACK1 proteins of Penaeus. The results of tissue expression and spatiotemporal expression showed that it was significantly increased in the II and IV stages, but had a significant tissue specificity in the hepatopancreas, spermary, and muscle tissues of E. carinicauda, adult stage. Compared to the control, EcRACK1 was significantly induced in E. carinicauda zoea larvae exposed to Aroclor 1254 for 6, 10, 20, and 30 d (p < 0.05). These results suggested that EcRACK1 may play an important role in the larval development and environmental defense of E. carinicauda.

WSB1/2 target chromatin-bound lysine-methylated RelA for proteasomal degradation and NF-κB termination.

Proteasome-mediated degradation of chromatin-bound NF-κB is critical in terminating the transcription of pro-inflammatory genes and can be triggered by Set9-mediated lysine methylation of the RelA subunit. However, the E3 ligase targeting methylated RelA remains unknown. Here, we find that two structurally similar substrate-recognizing components of Cullin-RING E3 ligases, WSB1 and WSB2, can recognize chromatin-bound methylated RelA for polyubiquitination and proteasomal degradation. We showed that WSB1/2 negatively regulated a subset of NF-κB target genes via associating with chromatin where they targeted methylated RelA for ubiquitination, facilitating the termination of NF-κB-dependent transcription. WSB1/2 specifically interacted with methylated lysines (K) 314 and 315 of RelA via their N-terminal WD-40 repeat (WDR) domains, thereby promoting ubiquitination of RelA. Computational modeling further revealed that a conserved aspartic acid (D) at position 158 within the WDR domain of WSB2 coordinates K314/K315 of RelA, with a higher affinity when either of the lysines is methylated. Mutation of D158 abolished WSB2's ability to bind to and promote ubiquitination of methylated RelA. Together, our study identifies a novel function and the underlying mechanism for WSB1/2 in degrading chromatin-bound methylated RelA and preventing sustained NF-κB activation, providing potential new targets for therapeutic intervention of NF-κB-mediated inflammatory diseases.

Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis

β-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain iron accumulation. Mutations in the WD repeat domain 45 (WDR45) gene encoding WIPI4 lead to loss of function in BPAN but the cellular mechanisms of how these trigger pathology are unclear. The prevailing view in the literature is that BPAN is simply the consequence of autophagy deficiency given that WIPI4 functions in this degradation pathway. However, our data indicate that WIPI4 depletion causes ferroptosis—a type of cell death induced by lipid peroxidation—via an autophagy-independent mechanism, as demonstrated both in cell culture and in zebrafish. WIPI4 depletion increases ATG2A localization at endoplasmic reticulum–mitochondrial contact sites, which enhances phosphatidylserine import into mitochondria. This results in increased mitochondrial synthesis of phosphatidylethanolamine, a major lipid prone to peroxidation, thus enabling ferroptosis. This mechanism has minimal overlap with classical ferroptosis stimuli but provides insights into the causes of neurodegeneration in BPAN and may provide clues for therapeutic strategies.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Deciphering the genetic landscape of seedling drought stress tolerance in wheat (Triticum aestivum L.) through genome-wide association studies.

Wheat is an important cereal crop constrained by several biotic and abiotic stresses including drought stress. Understating the effect of drought stress and the genetic basis of stress tolerance is important to develop drought resilient, high-yielding wheat cultivars. In this study, we investigated the effects of drought stress on seedling characteristics in an association panel consisting of 198 germplasm lines. Our findings revealed that drought stress had a detrimental effect on all the seedling characteristics under investigation with a maximum effect on shoot length (50.94% reduction) and the minimum effect on germination percentage (7.9% reduction). To gain a deeper understanding, we conducted a genome-wide association analysis using 12,511 single nucleotide polymorphisms (SNPs), which led to the identification of 39 marker-trait associations (MTAs). Of these 39 MTAs, 13 were particularly noteworthy as they accounted for >10% of the phenotypic variance with a LOD score >5. These high-confidence MTAs were further utilized to extract 216 candidate gene (CGs) models within 1 Mb regions. Gene annotation and functional characterization identified 83 CGs with functional relevance to drought stress. These genes encoded the WD40 repeat domain, Myb/SANT-like domain, WSD1-like domain, BTB/POZ domain, Protein kinase domain, Cytochrome P450, Leucine-rich repeat domain superfamily, BURP domain, Calmodulin-binding protein60, Ubiquitin-like domain, etc. Findings from this study hold significant promise for wheat breeders as they provide direct assistance in selecting lines harboring favorable alleles for improved drought stress tolerance. Additionally, the identified SNPs and CGs will enable marker-assisted selection of potential genomic regions associated with enhanced drought stress tolerance in wheat.

Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.

Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction, and ubiquitination assay confirmed the presence of a functional Skp1-Cullin-1-Fbox (PfSCF) complex, comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1, and calcyclin binding protein. Immunoprecipitation, sequence analysis, and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specificity factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. The protein levels of several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were significantly altered upon PfCullin-2 depletion, which likely reflects association of PfCRL4 with multiple pathways. PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had a significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. We demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity.

WSB1, as an E3 ligase, restrains myocardial ischemia–reperfusion injury by activating β-catenin signaling via promoting GSK3β ubiquitination

BackgroundReperfusion is the most effective strategy for myocardial infarct, but induces additional injury. WD repeat and SOCS box containing protein 1 (WSB1) plays a protective role in ischemic cells. This study aims to investigate the effects of WSB1 on myocardial ischemia–reperfusion (IR) injury.MethodsThe myocardial IR was induced by left anterior descending (LAD) ligation for 45 min and subsequent reperfusion. The overexpression of WSB1 was mediated by tail vein injection of AAV9 loaded with WSB1 encoding sequence two weeks before IR surgery. H9c2 myocardial cells underwent oxygen-sugar deprivation/reperfusion (OGD/R) to mimic IR, and transfected with WSB1 overexpression or silencing plasmid to alter the expression of WSB1.ResultsWSB1 was found highly expressed in penumbra of myocardial IR rats, and the WSB1 overexpression relieved IR-induced cardio dysfunction, myocardial infarct and pathological damage, and cardiomyocyte death in penumbra. The ectopic expression of WSB1 in H9c2 myocardial cells mitigated OGD/R-caused apoptosis, and silencing of WSB1 exacerbated the apoptosis. In addition, WSB1 activated β-catenin signaling, which was deactivated under the ischemic condition. The co-immunoprecipitation results revealed that WSB1 mediated ubiquitination and degradation of glycogen synthase kinase 3 beta (GSK3β) as an E3 ligase in myocardial cells. The effects of WSB1 on myocardial cells under ischemic conditions were abolished by an inhibitor of β-catenin signaling.ConclusionWSB1 activated β-catenin pathway by promoting the ubiquitination of GSK3β, and restrained IR-induced myocardial injury. These findings might provide novel insights for clinical treatment of myocardial ischemic patients.

MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells.

Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear. In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression. The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation. This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

The spectrum of neurological presentation in individuals affected by TBL1XR1 gene defects

BackgroundTBL1XR1 encodes a F-box-like/WD40 repeat-containing protein that plays a role in transcription mediated by nuclear receptors and is a known genetic cause of neurodevelopmental disease of childhood (OMIM# 608628). Yet the developmental trajectory and progression of neurologic symptoms over time remains poorly understood.MethodsWe developed and distributed a survey to two closed Facebook groups devoted to families of patients with TBL1XR1-related disorder. The survey consisted of 14 subsections focused upon the developmental trajectories of cognitive, behavioral, motor, and other neurological abnormalities. Data were collected and managed using REDCap electronic data capture tools.ResultsCaregivers of 41 patients with a TBL1XR1-related disorder completed the cross-sectional survey. All reported variants affecting a single amino acid, including missense mutations and in-frame deletions, were found in the WD40 repeat regions of Tbl1xr1. These are domains considered important for protein–protein interactions that may plausibly underlie disease pathology. The majority of patients were diagnosed with a neurologic condition before they received their genetic diagnosis. Language appeared most significantly affected with only a minority of the cohort achieving more advanced milestones in this domain.ConclusionTBL1XR1-related disorder encompasses a spectrum of clinical presentations, marked by early developmental delay ranging in severity, with a subset of patients experiencing developmental regression in later childhood.

Role of the leucine-rich repeat protein kinase 2 C-terminal tail in domain cross-talk.

Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Cancer-associated fibroblast exosomes promote prostate cancer metastasis through miR-500a-3p/FBXW7/HSF1 axis under hypoxic microenvironment.

Metastasis is the main cause of deaths in prostate cancer (PCa). However, the exact mechanisms underlying PCa metastasis are not fully understood. In this study, we discovered pronounced hypoxia in primary lesions of metastatic PCa(mPCa). The exosomes secreted by cancer-associated fibroblasts (CAFs) under hypoxic conditions significantly enhance PCa metastasis both in vitro and in vivo. Through miRNA sequencing and reverse transcription quantitative PCR (RT-qPCR), we found that hypoxia elevated miR-500a-3p levels in CAFs exosomes. Subsequent RT-qPCR, western blotting, and dual luciferase reporter assays identified F-box and WD repeat domain-containing 7(FBXW7) as a target of miR-500a-3p. In addition, immunohistochemistry revealed that FBXW7 expression decreased with the progression of PCa, while heat shock transcription factor 1(HSF1) expression increased. Introducing an FBXW7 plasmid into PCa cells reduced their metastatic potential and significantly lowered HSF1 expression. These findings suggest that CAFs exosomes drive PCa metastasis via the miR-500a-3p/FBXW7/HSF1 axis in a hypoxic microenvironment. Targeting either hypoxia or exosomal miR-500a-3p could be a promising strategy for PCa management.