Zinc Finger Research Articles

Targeting ZC3H11A elicits immunogenic cancer cell death through augmentation of antigen presentation and interferon response

Zinc finger CCCH containing 11A (ZC3H11A) is a stress-induced protein that is upregulated in various conditions, such as heat shock and virus infection. It has also been reported to be upregulated in certain cancers. The aim of this study was to evaluate the feasibility of targeting ZC3H11A as a therapeutic approach for cancer treatment, using nuclease-resistant, affinity-enhanced antisense oligonucleotide (ASO). An ASO targeting ZC3H11A was validated and evaluated in vitro and in the B16 melanoma model in vivo. Antigen presentation, interferon response, cell proliferation and apoptosis were transcriptionally affected. These findings were validated on the protein level by upregulation of major histocompatibility complex I (MHC-I), an increased secretion of interferon-β (IFN-β), and induction of apoptosis observed as upregulation of caspases and annexin V. Immunogenic features of the induced apoptosis were evidenced by surface exposure of calreticulin (CRT) and the secretion of ATP leading to enhanced dendritic cell (DC) phagocytosis, maturation, and activation. Treatment with the ZC3H11A-targeted ASO had limited efficacy in vivo while constitutive lentiviral shRNA knockdown of ZC3H11A in murine B16 melanoma cells, and human Hela cells led to reduced tumor growth with prolonged survival of mice, validating ZC3H11A as a relevant target for cancer therapy.

Jujuboside A Regulates Calcium Homeostasis and Structural Plasticity to Alleviate Depression-Like Behavior via Shh Signaling in Immature Neurons.

Depression, a leading cause of disability worldwide, is characterized by dysfunction of immature neurons, resulting in dysregulated calcium homeostasis and impaired structural plasticity. Jujuboside A (JuA), a biologically active compound derived from Semen Ziziphi Spinosae, has demonstrated anti-anxiety and anti-insomnia properties. Recent studies suggest that JuA may be a promising antidepressant, but its underlying mechanisms remain unclear. Sprague-Dawley rats were subjected to chronic unpredictable mild stress (CUMS) to induce a depression model. JuA (12.5 mg/kg, 25 mg/kg, 50 mg/kg) was administered orally for 4weeks. Emotional and cognitive function were assessed. Monoamine neurotransmitter levels were measured using enzyme-linked immunosorbent assay (ELISA). The number of immature neurons and calcium homeostasis were evaluated by immunofluorescence. Western blotting and immunofluorescence were employed to detect the expression of Sonic hedgehog (Shh) signaling proteins. Additionally, lentiviral vector expressing Shh shRNA (LV-Shh-RNAi) were infused intracerebrally to investigate the role of Shh in JuA's antidepressant effects. JuA significantly ameliorated depressive-like behavior and cognitive dysfunction in CUMS rats, increased monoamine neurotransmitter levels in serum and hippocampal tissue, reduced the number of BrdU/DCX (bromodeoxyuridine/doublecortin)-positive immature neurons, and attenuated calcium ion (Ca2+) concentration and Ca2+/calmodulin-dependent protein kinase II (CaMKII) levels in immature neurons. JuA also markedly elevated synaptic density and prominence complexity, upregulated Shh, Gli family zinc finger 1 and 2 (Gli1/2), synaptophysin (Syn) and postsynaptic density protein-95 (PSD-95) expression in the ventral dentate gyrus (vDG). However, knockdown of Shh in the vDG counteracted JuA's therapeutic effects. These findings collectively suggest that JuA improves depressive-like behavior in CUMS rats by modulating calcium homeostasis and synaptic structural plasticity in immature neurons through the Shh signaling pathway.

Evaluation of Potential Roles of Zinc Finger Homeobox 3 (Zfhx3) Expressed in Chondrocytes and Osteoblasts on Skeletal Growth in Mice.

Bone formation is tightly modulated by genetically encoded molecular proteins that interact to regulate cellular differentiation and secretion of bony matrix. Many transcription factors are known to coordinate these events by controlling gene transcription within networks. However, not all factors involved are known. Here, we identified a novel function forZinc Finger Homeobox 3(Zfhx3), a gene encoding a transcription factor, as a regulator of bone metabolism. We knocked outZfhx3conditionally in mice in either chondrocytes or osteoblasts and characterized their bones by micro-CT in 12-week-old mice. We observed a negative effect in linear bone growth in both knockout mice but reduced bone mass only in mice withZfhx3deleted in osteoblasts. Loss of Zfhx3 expression in osteoblasts affected trabecular bone mass in femurs and vertebrae in both sexes but influenced cortical bone volume fraction only in females. Moreover, transcriptional analysis of femoral bones in osteoblastZfhx3conditional knockout mice revealed a reduced expression of osteoblast genes, and histological evaluation of trabecular bones suggests that Zfhx3 causes changes in bone formation and not resorption. The loss of Zfhx3 causes reductions in trabecular bone area and osteoid volume, but no changes in the expression of osteoclast differentiation markers or number of TRAP stained osteoclasts. These studies introduce Zfhx3 as a relevant factor toward understanding gene regulatory networks that control bone formation and development of peak bone mass.

Comprehensive Annotation and Expression Profiling of C2H2 Zinc Finger Transcription Factors across Chicken Tissues.

As the most abundant class of transcription factors in eukaryotes, C2H2-type zinc finger proteins (C2H2-ZFPs) play critical roles in various biological processes. Despite being extensively studied in mammals, C2H2-ZFPs remain poorly characterized in birds. Recent accumulation of multi-omics data for chicken enables the genome-wide investigation of C2H2-ZFPs in birds. The purpose of this study is to reveal the genomic occurrence and evolutionary signature of chicken C2H2-ZFPs, and further depict their expression profiles across diverse chicken tissues. Here, we annotated 301 C2H2-ZFPs in chicken genome, which are associated with different effector domains, including KRAB, BTB, HOMEO, PHD, SCAN, and SET. Among them, most KRAB-ZFPs lack orthologues in mammals and tend to form clusters by duplication, supporting their fast evolution in chicken. We also annotated a unique and previously unidentified SCAN-ZFP, which is lineage-specific and highly expressed in ovary and testis. By integrating 101 RNA-seq datasets for 32 tissues, we found that most C2H2-ZFPs have tissue-specific expression. Particularly, 74 C2H2-ZFPs-including 27 KRAB-ZFPs-show blastoderm-enriched expression, indicating their association with early embryo development. Overall, this study performs comprehensive annotation and expression profiling of C2H2 ZFPs in diverse chicken tissues, which gives new insights into the evolution and potential function of C2H2-ZFPs in avian species.

SOX4-BMI1 axis promotes non-small cell lung cancer progression and facilitates angiogenesis by suppressing ZNF24.

The incidence of lung cancer has become the highest among all cancer types globally, also standing as a leading cause of cancer-related deaths. Lung cancer is broadly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the latter accounting for 85% of total cases. SRY-box transcription factor 4 (SOX4), a crucial transcription factor, has been found to play a key role in the development of various cancers. However, the association between SOX4 and NSCLC is still unclear. This study investigated the clinical relevance of SOX4 and its potential mechanisms in the progression of NSCLC. Analysis of our NSCLC patient cohort revealed a significant increase in SOX4 levels in cancerous tissues, indicating its role as an independent prognostic indicator for NSCLC. In vitro experiments demonstrated that elevated SOX4 expression facilitated NSCLC cell migration, invasion, and EMT. Functionally, SOX4 drives NSCLC progression by enhancing the transcription and expression of B-cell-specific moloney leukemia virus insertion site 1 (BMI1). The oncogenic impact of SOX4-induced BMI1 expression on NSCLC advancement was validated through both in vivo and in vitro studies. In addition, our findings showed that BMI1 promoted the ubiquitination of histone H2A (H2Aub), leading to decreased zinc finger protein 24 (ZNF24) expression, which subsequently triggered vascular endothelial growth factor A (VEGF-A) secretion in NSCLC cells, thereby promoting NSCLC angiogenesis. Moreover, we evaluated the therapeutic potential of a BMI1 inhibitor in combination with Bevacizumab for NSCLC treatment using orthotopic models. The data presented in our study reveal a previously unrecognized role of the SOX4-BMI1 axis in promoting NSCLC progression and angiogenesis. This research significantly contributes to our knowledge of the interplay between SOX4 and BMI1 in NSCLC, potentially paving the way for the development of targeted therapies for this disease.

Upregulation of Apoptosis Related Genes in Clinically Normal Tongue Contralateral to Squamous Cell Carcinoma of the Oral Tongue, an Effort to Maintain Tissue Homeostasis

PurposeThe field cancerization concept indicates the presence of pre-cancerous changes in clinically normal tissue surrounding the tumor. In squamous cell carcinoma of the oral tongue (SCCOT) which is infrequently linked to human papillomavirus infection, we have previously reported that clinically normal tongue contralateral to tumor (NTCT) is molecularly abnormal. Here, combining our transcriptomic and genomic data, we aimed to investigate the contribution of molecular changes in NTCT to cancer development.MethodsMicroarray gene expression data of 14 healthy controls, 23 NTCT and 29 SCCOT samples were investigated to characterize transcriptional profiles in NTCT. Whole exome sequencing and RNA-sequencing data of paired NTCT and tumor samples from 15 SCCOT patients were used to study correlation between copy number variation and differential gene expression.ResultsUsing supervised multivariate partial least squares discriminant analysis, a total of 61 mRNAs that distinguish NTCT from healthy tongue were selected. Functional enrichment analysis of the 22 upregulated genes showed increased “positive regulation of nitrogen compound metabolic process” in NTCT. All 12 genes involved in this process have roles in apoptosis (anti- and/or pro-apoptotic). Compared to healthy controls, Zinc Finger Protein 395 (ZNF395), a pro-apoptotic tumor suppressor located on chromosome 8p, was the only gene showing increased mRNA level in NTCT whereas decreased in SCCOT. Given the frequent loss of chromosome 8p in SCCOT, the impact of ZNF395 copy number variation on gene expression was further examined, revealing a positive correlation between copy number and mRNA level (correlation coefficient = 0.572, p < 0.001).ConclusionNTCT is susceptible to malignant transformation, where tissue homeostasis is maintained at least partly through regulation of apoptosis. Loss of the pro-apoptotic gene ZNF395 could thus initiate cancer development.

Structural insights into the recognition of purine-pyrimidine dinucleotide repeats by zinc finger protein ZBTB43.

Purine-pyrimidine repeats (PPRs) can form left-handed Z-form DNA and induce DNA double-strand breaks (DSBs), posing a risk for genomic rearrangements and cancer. The zinc finger (ZF) and BTB domain-containing protein 43 (ZBTB43) is a transcription factor containing two Cys2-His2 (C2H2) and one C3H1 zinc fingers and plays a crucial role in maintaining genomic and epigenomic integrity by converting mutagenic Z-form PPRs to the B-form in prospermatogonia. Despite its importance, the molecular mechanism underlying the recognition of PPRs by ZBTB43 remains elusive. In this study, we determined the X-ray crystal structure of the ZBTB43 ZF1-3 in complex with the B-form DNA containing the CA repeats sequence. The structure reveals that ZF1 and ZF2 primarily recognize the CACA sequence through specific hydrogen-bonding and van der Waals contacts via a quadruple center involving Arg389, Met411, His413, and His414. These interactions were further validated by fluorescence-based DNA-binding assays using mutated ZBTB43 variants. Our structural investigation provides valuable insights into the recognition mechanism of PPRs by ZBTB43 and suggests a potential role for ZBTB43 in the transformation of Z-DNA to B-DNA, contributing to the maintenance of genomic stability.

METTL3-mediated m6A modification of ZNF384 promotes hepatocellular carcinoma progression by transcriptionally activating ACSM1.

Hepatocellular carcinoma (HCC) is a lethal disease with a high mortality rate, and its development is influenced by various molecular mechanisms. Zinc finger protein 384 (ZNF384) has been reported to be involved in the progression of several cancers; however, its role in HCC remains elusive. mRNA expression levels were analyzed by quantitative real-time polymerase chain reaction, while western blotting and immunohistochemistry were performed to validate protein expression. Cell proliferation, apoptosis, and metabolic activities were examined using clonogenicity, flow cytometry, and specific assay kits. A xenograft mouse model was employed to assess the impact of acyl-CoA synthetase medium-chain family member 1 (ACSM1) depletion on HCC cell malignancy in vivo. Chromatin immunoprecipitation assay and dual-luciferase reporter assay were conducted to explore the association between ZNF384 and ACSM1. We found that ACSM1 and ZNF384 were significantly upregulated in HCC tissues and cells when compared with normal liver tissues and human liver immortalized cells. Knockdown of ACSM1 inhibited HCC cell proliferation and glucose metabolism and induced cell apoptosis. Furthermore, ACSM1 depletion suppressed the malignant progression of HCC cells in vivo. Our data indicated that ZNF384 transcriptionally activated ACSM1 in HCC cells. Overexpression of ACSM1 reversed the inhibitory effect of ZNF384 depletion on HCC cell malignancy. Further, methyltransferase-like 3 (METTL3) stabilized ZNF384 mRNA through m6A methylation. METTL3-mediated m6A modification of ZNF384 contributed to the progression of HCC by transcriptionally activating ACSM1. This finding suggests potential therapeutic targets for this devastating disease.

Expanded Gene Regulatory Network Reveals Potential Light-Responsive Transcription Factors and Target Genes in Cordyceps militaris.

Cordyceps militaris, a fungus widely used in traditional Chinese medicine and pharmacology, is recognized for its abundant bioactive compounds, including cordycepin and carotenoids. The growth, development, and metabolite production in various fungi are influenced by the complex interactions between regulatory cascades and light-signaling pathways. However, the mechanisms of gene regulation in response to light exposure in C. militaris remain largely unexplored. This study aimed to identify light-responsive genes and potential transcription factors (TFs) in C. militaris through an integrative transcriptome analysis. To achieve this, we reconstructed an expanded gene regulatory network (eGRN) comprising 507 TFs and 8662 regulated genes using both interolog-based and homolog-based methods to build the protein-protein interaction network. Aspergillus nidulans and Neurospora crassa were chosen as templates due to their relevance as fungal models and the extensive study of their light-responsive mechanisms. By utilizing the eGRN as a framework for comparing transcriptomic responses between light-exposure and dark conditions, we identified five key TFs-homeobox TF (CCM_07504), FlbC (CCM_04849), FlbB (CCM_01128), C6 zinc finger TF (CCM_05172), and mcrA (CCM_06477)-along with ten regulated genes within the light-responsive subnetwork. These TFs and regulated genes are likely crucial for the growth, development, and secondary metabolite production in C. militaris. Moreover, molecular docking analysis revealed that two novel TFs, CCM_05727 and CCM_06992, exhibit strong binding affinities and favorable docking scores with the primary light-responsive protein CmWC-1, suggesting their potential roles in light signaling pathways. This information provides an important functional interactive network for future studies on global transcriptional regulation in C. militaris and related fungi.

Transcription factor SlSTOP1 regulates Small Auxin-Up RNA Genes for tomato root elongation under aluminum stress.

Aluminum (Al) stress, a prevalent constraint in acidic soils, inhibits plant growth by inhibiting root elongation through restricted cell expansion. The molecular mechanisms of Al-induced root inhibition, however, are not fully understood. This study aimed to elucidate the role of Small Auxin-up RNAs (SlSAURs), which function downstream of the key Al stress-responsive transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (SlSTOP1) and its enhancer STOP1-INTERACTING ZINC-FINGER PROTEIN 1 (SlSZP1), in modulating root elongation under Al stress in tomato (Solanum lycopersicum). Our findings demonstrated that tomato lines with knocked out SlSAURs exhibited shorter root lengths when subjected to Al stress. Further investigation into the underlying mechanisms revealed that SlSAURs interact with Type 2C Protein Phosphatases (SlPP2Cs), specifically D-clade Type 2C Protein Phosphatases (SlPP2C.Ds). This interaction was pivotal as it suppresses the phosphatase activity, leading to the degradation of SlPP2C.D's inhibitory effect on plasma membrane H+-ATPase. Consequently, this promoted cell expansion and root elongation under Al stress. These findings increase our understanding of the molecular mechanisms by which Al ions modulate root elongation. The discovery of the SlSAUR-SlPP2C.D interaction and its impact on H+-ATPase activity also provides a perspective on the adaptive strategies employed by plants to cope with Al toxicity, which may lead to the development of tomato cultivars with enhanced Al stress tolerance, thereby improving crop productivity in acidic soils.

Identification of dystrophin Dp71dΔ71-associated proteins in PC12 cells by quantitative proteomics

Dystrophin Dp71 is essential for the development of the nervous system. Its alteration is associated with intellectual disability. Different Dp71 isoforms are generated by alternative splicing; however, their functions have not been fully described. Here, we identified Dp71dΔ71-associated proteins to understand the complex functions. PC12 cells, stably transfected with pTRE2pur-Myc/Dp71dΔ71 or pTRE2pur-Myc empty vector (EV), were analyzed by immunoprecipitation followed with quantitative proteomics with data-independent acquisition and ion mobility separation. We used the Top3 method to quantify absolutely every protein detected. A total of 106 proteins were quantified with Progenesis QI software and the database UP000002494. Seven new proteins associated with Dp71dΔ71 were selected with at least 2-fold quantity between immunoprecipitated proteins of PC12-Myc/Dp71dΔ71 versus PC12-EV cells. These results revealed new proteins that interact with Dp71dΔ71, including β-Tubulin, S-adenosylmethionine synthase isoform type-2, adapter molecule crk, helicase with zinc finger 2, WD repeat domain 93, cyclin-L2 and myosin-10, which are related to cell migration and/or cell growth. The results lay the foundation for future research on the relationship between these proteins and Dp71 isoforms.

Myricanol represses renal fibrosis by activating TFAM and ZNRF1 to inhibit tubular epithelial cells ferroptosis

BackgroundMitochondrial dysfunction induces ferroptosis in renal tubular epithelial cells (TECs). Studies have shown that myricanol maintains muscle cell function by enhancing mitochondrial energy metabolism. HypothesisMyricanol delays renal fibrosis by maintaining mitochondrial integrity and inhibiting ferroptosis in TECs. MethodsMice kidney lacking mitochondrial transcription factor A (TFAM), blood specimens, or pathological sections of renal tissue from patients with renal failure were used to explore the relationship between mitochondrial and renal functions. Erastin induced-TECs ferroptosis was used to study the potential mechanism by which TFAM regulates renal fibrosis. Chronic kidney disease (CKD) mice were utilized to explore the anti-fibrotic effects of myricanol. ResultsThe number of mitochondria and TFAM expression were decreased in human blood samples and pathological sections. Renal TFAM-deficient mice exhibited abnormalities in renal function, including ferroptosis and fibrosis. Ferrostatin-1 significantly inhibited renal fibrosis by preventing TECs ferroptosis. Transcriptional sequencing results indicated that zinc and ring finger 1 (ZNRF1) were important downstream genes of TFAM that regulate ferroptosis. We demonstrated that TFAM deficiency and ferroptosis, which destroyed interaction between ZNRF1 and the iron transport-related protein lipocalin-2 (LCN2), but myricanol clould reverse this effect. Overexpression of ZNRF1 efficiently maintained mitochondrial integrity and inhibited renal fibrosis. Myricanol ameliorated transforming growth factor β1-induced mitochondrial impairment. We firstly confirmed that myricanol efficiently improved renal function and suppresses fibrosis in CKD mice. ConclusionsMyricanol efficiently inhibit fibrosis through activating TFAM to stimulate the interaction between ZNRF1 and LCN2.

Epigenetic reader ZMYND11 noncanonical function restricts HNRNPA1-mediated stress granule formation and oncogenic activity

Epigenetic readers frequently affect gene regulation, correlate with disease prognosis, and hold significant potential as therapeutic targets for cancer. Zinc finger MYND-type containing 11 (ZMYND11) is notably recognized for reading the epigenetic marker H3.3K36me3; however, its broader functions and mechanisms of action in cancer remain underexplored. Here, we report that ZMYND11 downregulation is prevalent across various cancers and profoundly correlates with poorer outcomes in prostate cancer patients. Depletion of ZMYND11 promotes tumor cell growth, migration, and invasion in vitro, as well as tumor formation and metastasis in vivo. Mechanistically, we discover that ZMYND11 exhibits tumor suppressive roles by recognizing arginine-194-methylated HNRNPA1 dependent on its MYND domain, thereby retaining HNRNPA1 in the nucleus and preventing the formation of stress granules in the cytoplasm. Furthermore, ZMYND11 counteracts the HNRNPA1-driven increase in the PKM2/PKM1 ratio, thus mitigating the aggressive tumor phenotype promoted by PKM2. Remarkably, ZMYND11 recognition of HNRNPA1 can be disrupted by pharmaceutical inhibition of the arginine methyltransferase PRMT5. Tumors with low ZMYND11 expression show sensitivity to PRMT5 inhibitors. Taken together, our findings uncover a previously unexplored noncanonical role of ZMYND11 as a nonhistone methylation reader and underscore the critical importance of arginine methylation in the ZMYND11-HNRNPA1 interaction for restraining tumor progression, thereby proposing novel therapeutic targets and potential biomarkers for cancer treatment.

ZDHHC7-mediated S-palmitoylation of ATG16L1 facilitates LC3 lipidation and autophagosome formation

ABSTRACT Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12–ATG5 conjugate to form an ATG12–ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy. Abbreviation: ABE: acyl-biotin exchange; ATG: autophagy related; Baf-A1: bafilomycin A1; 2-BP: 2-bromopalmitate; CCD: coiled‐coil domain; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle’s balanced salt solution; HAM: hydroxylamine; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NP-40: Nonidet P-40; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex I; PTM: post-translational modification; RAB33B: RAB33B, member RAS oncogene family; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; WD: tryptophan and aspartic acid; WIPI2B: WD repeat domain, phosphoinositide interacting 2B; WT: wild-type; ZDHHC: zinc finger DHHC-type palmitoyltransferase.

An Anti-Invasive Role for Mdmx through the RhoA GTPase under the Control of the NEDD8 Pathway.

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.

OsHRZ1 negatively regulates rice resistant to Magnaporthe oryzae infection by targeting OsVOZ2.

Rice blast disease caused by Magnaporthe oryzae significantly reduces yield production. Blast resistance is closely associated with iron (Fe) status, but the mechanistic basis linking iron status to immune function in rice remains largely unknown. Here, iron-binding haemerythrin RING ubiquitin ligases OsHRZ1 was confirmed to play key roles in iron-mediated rice blast resistance. The expression of OsHRZ1 was suppressed by M. oryzae inoculation and high iron treatment. Both mutants of OsHRZ1 enhanced rice resistance to M. oryzae. OsPR1a was up-regulated in OsHRZ1 mutants. Yeast two-hybrid, bimolecular fluorescence complementation, and Co-IP assay results indicated that OsHRZ1 interacts with Vascular Plant One Zinc Finger 2 (OsVOZ2) in the nucleus. Additionally, the vitro ubiquitination assay indicated that OsHRZ1 can ubiquitinate OsVOZ2 and mediate the degradation of OsVOZ2. The mutants of OsVOZ2 showed reduced resistance to M. oryzae and down-regulated the expression of OsPR1a. Yeast one-hybrid, EMSA, and dual-luciferase reporter assay results indicated that OsVOZ2 directly binds to the promoter of OsPR1a, activating its expression. In summary, OsHRZ1 plays an important role in rice disease resistance by mediated degradation of OsVOZ2 thus shaping PR gene expression dynamics in rice cells. This highlights an important link between iron signaling and rice pathogen defenses.

USP5 promotes tumor progression by stabilizing SLUG in bladder cancer.

Bladder cancer ranks as the second most prevalent urology malignancy globally. Invasive metastasis is a significant contributor to mortality among patients with bladder cancer, yet the underlying mechanisms remain elusive. Deubiquitinases are pivotal in carcinogenesis, with USP5 implicated in the malignant progression of hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer. The present study assessed the role and mechanism of ubiquitin-specific proteinase 5 (USP5) in the malignant progression of bladder cancer. The association between USP5 expression and bladder cancer prognosis and stage was analyzed using The Cancer Genome Atlas database. Moreover, to elucidate the role of USP5 in bladder cancer, USP5 overexpression and knockdown cell lines were established using T24 cells. Cell viability, proliferation and migration were assessed using Cell Counting Kit-8, Transwell and scratch assays, respectively. Cyclohexanamide was used to evaluate the effect of USP5 expression on Snail family zinc finger 2 (SLUG) stability. Immunoprecipitation and immunofluorescence co-localization were utilized to probe the interaction between USP5 and SLUG. Changes in mRNA and protein levels were assessed using reverse transcription-quantitative PCR and western blotting, respectively. The results revealed that patients with bladder cancer with high USP5 expression had significantly shorter survival (P<0.05) and a higher clinicopathologic stage (P<0.05) than those with low USP5 expression. T24 cells overexpressing USP5 demonstrated significantly increased proliferation (P<0.05), invasion (P<0.05) and expression of epithelial-mesenchymal transition markers (P<0.05); whereas T24 cells with knocked-down USP5 expression revealed significantly reduced proliferation (P<0.05), invasion (P<0.05) and epithelial-mesenchymal transition markers (P<0.05). Immunoprecipitation experiments demonstrated the binding of USP5 to SLUG in bladder cancer cells, with further analysis revealing that USP5 upregulated protein levels of SLUG by inhibiting its ubiquitination. Furthermore, the treatment of bladder cancer cells with Degrasyn, a USP5 inhibitor, was associated with a significant inhibition of the proliferation (P<0.05) and invasion (P<0.05) of T24 cells. In conclusion, the findings of the present study underscore the role of USP5 in promoting the malignant progression of bladder cancer through the stabilization of SLUG. Targeting USP5 holds promise as a strategy for inhibiting bladder cancer progression.

Genome-Wide Characterization of the INDETERMINATE DOMAIN (IDD) Zinc Finger Gene Family in Solanum lycopersicum and the Functional Analysis of SlIDD15 in Shoot Gravitropism.

The plant-specific IDD transcription factors (TFs) are vital for regulating plant growth and developmental processes. However, the characteristics and biological roles of the IDD gene family in tomato (Solanum lycopersicum) are still largely unexplored. In this study, 17 SlIDD genes were identified in the tomato genome and classified into seven subgroups according to the evolutionary relationships of IDD proteins. Analysis of exon-intron structures and conserved motifs reflected the evolutionary conservation of SlIDDs in tomato. Collinearity analysis revealed that segmental duplication promoted the expansion of the SlIDD family. Ka/Ks analysis indicated that SlIDD gene orthologs experienced predominantly purifying selection throughout evolution. The analysis of cis-acting elements revealed that the promoters of SlIDD genes contain numerous elements associated with light, plant hormones, and abiotic stresses. The RNA-seq data and qRT-PCR experimental results showed that the SlIDD genes exhibited tissue-specific expression. Additionally, Group A members from Arabidopsis thaliana and rice are known to play a role in regulating plant shoot gravitropism. QRT-PCR analysis confirmed that the expression level of SlIDD15 in Group A was high in the hypocotyls and stems. Subcellular localization demonstrated that the SlIDD15 protein was localized in the nucleus. Surprisingly, the loss-of-function of SlIDD15 by CRISPR/Cas9 gene editing technology did not display obvious gravitropic response defects, implying the existence of functional redundant factors within SlIDD15. Taken together, this study offers foundational insights into the tomato IDD gene family and serves as a valuable guide for exploring their molecular mechanisms in greater detail.

The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome.

Inherited deficiency of zinc finger NFX1-type containing 1 (ZNFX1), a dsRNA virus sensor, is associated with severe familial immunodeficiency, multisystem inflammatory disease, increased susceptibility to viruses, and early mortality. However, limited treatments for patients with pathological variants of ZNFX1 exist due to an incomplete understanding of the diseases resulting from ZNFX1 mutations. Here, we demonstrate that ZNFX1 specifically inhibits the activation of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome in response to NLRP3 activators both in vitro and in vivo. ZNFX1 retains NLRP3 in the cytoplasm and prevents its accumulation in the TGN38 + /TGN46+ vesicles in the resting state. Upon NLRP3 inflammasome activation, ZNFX1 is cleaved by caspase-1, establishing a feed-forward loop that promotes NLRP3 accumulation in the trans-Golgi network (TGN) and amplifies the activity of the downstream cascade. Expression of wild-type ZNFX1, but not of ZNFX1 with human pathogenic mutations, rescues the impairment of NLRP3 inflammasome inhibition. Our findings reveal a dual role of ZNFX1 in virus sensing and suppression of inflammation, which may become valuable for the development of treatments for ZNFX1 mutation-related diseases.

The joint toxicity effect of glyphosate and cadmium in a concentration-dependent manner on nematode Caenorhabditis elegans

The co-occurrence of glyphosate (GPS), a commonly used organophosphorus herbicide, and cadmium (Cd), a neurotoxic metal, in agricultural environments prompts concerns about their combined toxic effects on ecosystems. This study explores the combined effects of GPS and Cd on the model organism Caenorhabditis elegans (C. elegans), to understand their cumulative effects in organismal living environments. We investigated the interaction between GPS and Cd over 24 hours using a comprehensive approach that included a variety of toxicity endpoints as well as the novel Automated Recognition and Statistics Tool (NCLE) for body bend measurement. Our data show a concentration-dependent interplay in which antagonistic effects at lower concentrations reduce phenotypic damage while synergistic effects emerge at higher concentrations, particularly at GPS's LC50. Transcriptome analysis under antagonistic conditions revealed significant downregulation of Cd toxicity-related genes and identified Y22D7AL.16, which has a C2H2-type zinc finger domain, as a novel gene involved in metal stress response, implying an alternative Cd-resilience mechanism. The expression profile of this gene shows that it plays a larger role in both development and metal stress adaption. These findings highlight the complexities of compound pollutant interactions, emphasizing the importance of including such dynamics in environmental risk assessments and control techniques.