Nuclear Localization Research Articles

Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications

PurposesThe objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity. MethodsTwo lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene. ResultsOur findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6–8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0. ConclusionThese results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.

A glycine-rich nuclear effector VdCE51 of Verticillium dahliae suppresses plant immune responses by inhibiting the accumulation of GhTRXH2

Verticillium dahliae is an important soil-borne fungal pathogen that causes great yield losses in many cash crops. Effectors of this fungus are known to regulate plant immunity but the mechanism much remains unclear. A glycine-rich nuclear effector, VdCE51, was able to suppress immune responses in tobacco against Botrytis cinerea and Sclerotinia sclerotiorum. This effector was a required factor for full virulence of V. dahliae, and its nuclear localization was a requisite for suppressing plant immunity. The thioredoxin GhTRXH2, identified as a positive regulator of plant immunity, was a host target of VdCE51. Our findings show a virulence regulating mechanism whereby the secreted nuclear effector VdCE51 interferes with the transcription of PR genes, and the SA signaling pathway by inhibiting the accumulation of GhTRXH2, thus suppressing plant immunity.

High-dose methylprednisolone mediates YAP/TAZ-TEAD in vocal fold fibroblasts with macrophages.

The pro-fibrotic effects of glucocorticoids may lead to a suboptimal therapeutic response for vocal fold (VF) pathology. Targeting macrophage-fibroblast interactions is an interesting therapeutic strategy; macrophages alter their phenotype to mediate both inflammation and fibrosis. In the current study, we investigated concentration-dependent effects of methylprednisolone on the fibrotic response, with an emphasis on YAP/TAZ-TEAD signaling, and inflammatory gene expression in VF fibroblasts in physical contact with macrophages. We sought to provide foundational data to optimize therapeutic strategies for millions of patients with voice/laryngeal disease-related disability. Following induction of inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes, THP-1-derived macrophages were seeded onto HVOX vocal fold fibroblasts. Cells were co-cultured +/-0.3-3000nM methylprednisolone +/- 3µM verteporfin, a YAP/TAZ inhibitor. Inflammatory ( CXCL10 , TNF , PTGS2 ) and fibrotic genes ( ACTA2 , CCN2 , COL1A1 ) in fibroblasts were analyzed by real-time polymerase chain reaction after cell sorting. Ser211-phosphorylated glucocorticoid receptor (S211-pGR) was assessed by Western blotting. Nuclear localization of S211-pGR and YAP/TAZ was analyzed by immunocytochemistry. Methylprednisolone decreased TNF and PTGS2 in fibroblasts co-cultured with M(IFN/LPS) macrophages and increased ACTA2 and CCN2 in fibroblasts co-cultured with M(IFN/LPS) and M(TGF). Lower concentrations were required to decrease TNF and PTGS2 expression and to increase S211-pGR than to increase ACTA2 and CCN2 expression and nuclear localization of S211-pGR. Methylprednisolone also increased YAP/TAZ nuclear localization. Verteporfin attenuated upregulation of CCN2 , but not PTGS2 downregulation. High concentration methylprednisolone induced nuclear localization of S211-pGR and upregulated fibrotic genes mediated by YAP/TAZ activation.

Porphyromonas gingivalis virulence factors induce toxic effects in SH-SY5Y neuroblastoma cells: GRK5 modulation as a protective strategy

Periodontitis (PDS) is a chronic inflammatory disease initiated by a dysbiosis of oral pathogenic bacterial species, such as Porphyromonas gingivalis (Pg). These bacteria can penetrate the bloodstream, releasing various endo and exotoxins that fuel the infection, and stimulate toxic inflammation in different compartments, including the brain. However, the specific mechanisms by which PDS/Pg contribute to brain disorders, such as Alzheimer’s disease (AD), remain unclear. This study assessed the effects of Pg’s virulence factors - lipopolysaccharide (LPS-Pg) and gingipains (gps) K (Kgp) and Rgp - on SH-SY5Y cells. Our results demonstrated that LPS-Pg activated signaling through the Toll-like receptor (TLR)-2/4 induced a significant downregulation of G protein-coupled receptor kinase 5 (GRK5). Additionally, LPS-Pg stimulation resulted in a robust increase in Tau phosphorylation (pTau) and p53 levels, while causing a marked reduction in Bcl2 and increased cell death compared to unstimulated cells (Ns). LPS-Pg also elevated inducible nitric oxide synthase (iNOS) expression, leading to oxidative damage. In cells overexpressing GRK5 via Adenovirus, LPS-Pg failed to increase iNOS and pTau levels compared to GFP control cells. High GRK5 levels also prevented the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Furthermore, the overexpression of a GRK5 mutant form lacking the nuclear localization signal (ΔNLS) nearly abolished LPS-Pg induced p53 and iNOS upregulation. Finally, we tested whether Kgp and Rgp mediated similar effects and our data showed that both gps caused a marked downregulation of GRK5 leading to increased p53 and pTau levels.In conclusion, this study provides further insight into the toxic effects elicited by Pg in cells and suggests that preventing GRK5 deficiency may be a valid strategy to mitigate Pg-induced toxic effects (i.e. cell death, oxidative damage, and Tau hyperphosphorylation) in SH-SY5Y cells, which are typical molecular hallmarks of neurodegenerative disorders.

Lack of Nuclear Localization of the Creb3l1 Transcription Factor Causes Defects in Caudal Fin Bifurcation in Zebrafish Danio rerio.

The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription. We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

The C2H2 Transcription Factor Con7 Regulates Vegetative Growth, Cell Wall Integrity, Oxidative Stress, Asexual Sporulation, Appressorium and Hyphopodium Formation, and Pathogenicity in Colletotrichum graminicola and Colletotrichum siamense.

The Colletotrichum genus is listed as one of the top 10 important plant pathogens, causing significant economic losses worldwide. The C2H2 zinc finger protein serves as a crucial transcription factor regulating growth and development in fungi. In this study, we identified two C2H2 transcription factors, CgrCon7 and CsCon7, in Colletotrichum graminicola and Colletotrichum siamense, as the orthologs of Con7p in Magnaporthe oryzae. Both CgrCon7 and CsCon7 have a typical C2H2 zinc finger domain and exhibit visible nuclear localization. Disrupting Cgrcon7 or Cscon7 led to a decreased growth rate, changes in cell wall integrity, and low tolerance to H2O2. Moreover, the deletion of Cgrcon7 or Cscon7 dramatically decreased conidial production, and their knockout mutants also lost the ability to produce appressoria and hyphopodia. Pathogenicity assays displayed that deleting Cgrcon7 or Cscon7 resulted in a complete loss of virulence. Transcriptome analysis showed that CgrCon7 and CsCon7 were involved in regulating many genes related to ROS detoxification, chitin synthesis, and cell wall degradation, etc. In conclusion, CgrCon7 and CsCon7 act as master transcription factors coordinating vegetative growth, oxidative stress response, cell wall integrity, asexual sporulation, appressorium formation, and pathogenicity in C. graminicola and C. siamense.

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

The Spectrum of Renal "TFEopathies": Flipping the mTOR Switch in Renal Tumorigenesis.

The mammalian target of Rapamycin complex 1 (mTORC1) is a serine/threonine kinase that couples nutrient and growth factor signaling to the cellular control of metabolism and plays a fundamental role in aberrant proliferation in cancer. mTORC1 has previously been considered an "on/off" switch, capable of phosphorylating the entire pool of its substrates when activated. However, recent studies have indicated that mTORC1 may be active toward its canonical substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase (S6K), involved in mRNA translation and protein synthesis, and inactive toward TFEB and TFE3, transcription factors involved in the regulation of lysosome biogenesis, in several pathological contexts. Among these conditions are Birt-Hogg-Dubé syndrome (BHD) and, recently, tuberous sclerosis complex (TSC). Furthermore, increased TFEB and TFE3 nuclear localization in these syndromes, and in translocation renal cell carcinomas (tRCC), drives mTORC1 activity toward the canonical substrates, through the transcriptional activation of the Rag GTPases, thereby positioning TFEB and TFE3 upstream of mTORC1 activity toward 4EBP1 and S6K. The expanding importance of TFEB and TFE3 in the pathogenesis of these renal diseases warrants a novel clinical grouping that we term "TFEopathies." Currently, there are no therapeutic options directly targeting TFEB and TFE3, which represents a challenging and critically required avenue for cancer research.

The Non-Histone Protein FgNhp6 Is Involved in the Regulation of the Development, DON Biosynthesis, and Virulence of Fusarium graminearum.

Fusarium graminearum is the primary causative agent of Fusarium head blight (FHB), a devastating disease affecting cereals globally. The high-mobility group (HMG) of non-histone proteins constitutes vital architectural elements within chromatin, playing diverse roles in various biological processes in eukaryotic cells. Nonetheless, the specific functions of HMG proteins in F. graminearum have yet to be elucidated. Here, we identified 10 HMG proteins in F. graminearum and extensively characterized the biological roles of one HMGB protein, FgNhp6. We constructed the FgNhp6 deletion mutant and its complementary strains. With these strains, we confirmed the nuclear localization of FgNhp6 and discovered that the absence of FgNhp6 led to reduced radial growth accompanied by severe pigmentation defects, a significant reduction in conidial production, and a failure to produce perithecia. The ∆FgNhp6 mutant exhibited a markedly reduced pathogenicity on wheat coleoptiles and spikes, coupled with a significant increase in deoxynivalenol production. An RNA sequencing (RNA-seq) analysis indicated that FgNhp6 deletion influenced a wide array of metabolic pathways, particularly affecting several secondary metabolic pathways, such as sterol biosynthesis and aurofusarin biosynthesis. The findings of this study highlight the essential role of FgNhp6 in the regulation of the asexual and sexual reproduction, deoxynivalenol (DON) production, and pathogenicity of F. graminearum.

Kv1.3-induced hyperpolarization is required for efficient Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.

Germ line ERG haploinsufficiency defines a new syndrome with cytopenia and hematological malignancy predisposition

AbstractThe genomics era has facilitated the discovery of new genes that predispose individuals to bone marrow failure (BMF) and hematological malignancy (HM). We report the discovery of ETS-related gene (ERG), a novel, autosomal dominant BMF/HM predisposition gene. ERG is a highly constrained transcription factor that is critical for definitive hematopoiesis, stem cell function, and platelet maintenance. ERG colocalizes with other transcription factors, including RUNX family transcription factor 1 (RUNX1) and GATA binding protein 2 (GATA2), on promoters or enhancers of genes that orchestrate hematopoiesis. We identified a rare heterozygous ERG missense variant in 3 individuals with thrombocytopenia from 1 family and 14 additional ERG variants in unrelated individuals with BMF/HM, including 2 de novo cases and 3 truncating variants. Phenotypes associated with pathogenic germ line ERG variants included cytopenias (thrombocytopenia, neutropenia, and pancytopenia) and HMs (acute myeloid leukemia, myelodysplastic syndrome, and acute lymphoblastic leukemia) with onset before 40 years. Twenty ERG variants (19 missense and 1 truncating), including 3 missense population variants, were functionally characterized. Thirteen potentially pathogenic erythroblast transformation specific (ETS) domain missense variants displayed loss-of-function (LOF) characteristics, thereby disrupting transcriptional transactivation, DNA binding, and/or nuclear localization. Selected variants overexpressed in mouse fetal liver cells failed to drive myeloid differentiation and cytokine-independent growth in culture and to promote acute erythroleukemia when transplanted into mice, concordant with these being LOF variants. Four individuals displayed somatic genetic rescue by copy neutral loss of heterozygosity. Identification of predisposing germ line ERG variants has clinical implications for patient and family diagnoses, counseling, surveillance, and treatment strategies, including selection of bone marrow donors and cell or gene therapy.

Lipopolysaccharide-responsive beige-like anchor is involved in regulating NF-κB activation in B cells.

Lipopolysaccharide-responsive and beige-like anchor (LRBA) is a scaffolding protein that interacts with proteins such as CTLA-4 and PKA, the importance of which has been determined in various cell types, including T regulatory cells, B cells, and renal cells. LRBA deficiency is associated with an inborn error in immunity characterized by immunodeficiency and autoimmunity. In addition to defects in T regulatory cells, patients with LRBA deficiency also exhibit B cell defects, such as reduced cell number, low memory B cells, hypogammaglobulinemia, impaired B cell proliferation, and increased autophagy. Although Lrba-/- mice do not exhibit the immunodeficiency observed in humans, responses to B cell receptors (BCR) in B cells have not been explored. Therefore, a murine model is for elucidating the mechanism of Lrba mechanism in B cells. To compare and evaluate spleen-derived B cell responses to BCR crosslinking in C57BL6 Lrba-/- and Lrba+/+ mice. Spleen-derived B cells were obtained from 8 to 12-week-old mice. Subpopulations were determined by immunostaining and flow cytometry. BCR crosslinking was assessed by the F(ab')2 anti-μ chain. Activation, proliferation and viability assays were performed using flow cytometry and protein phosphorylation was evaluated by immunoblotting. The nuclear localization of p65 was determined using confocal microscopy. Nur77 expression was evaluated by Western blot. Lrba-/- B cells showed an activated phenotype and a decreased proportion of transitional 1 B cells, and both proliferation and survival were affected after BCR crosslinking in the Lrba-/- mice. The NF-κB pathway exhibited a basal activation status of several components, resulting in increased activation of p50, p65, and IκBα, basal p50 activation was reduced by the Plcγ2 inhibitor U73122. BCR crosslinking in Lrba-/ - B cells resulted in poor p50 phosphorylation and p65 nuclear localization. Increased levels of Nur77 were detected. These results indicate the importance of Lrba in controlling NF-κB activation driven by BCR. Basal activation of NF-κB could impact cellular processes, such as, activation, differentiation, proliferation, and maintenance of B cells after antigen encounter.

Proteome-scale characterisation of motif-based interactome rewiring by disease mutations

Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome.

Nuclear alpha-synuclein accelerates cell senescence and neurodegeneration

BackgroundThe progression of Parkinson’s disease (PD) is related to ageing. The accumulation of nuclear alpha-synuclein (α-syn) may accelerate the occurrence of neurodegenerative diseases, but its role in PD remains poorly understood.MethodsIn the present study, α-syn expression was specifically targeted to the nucleus by constructing an adeno-associated virus (AAV) vector in which a nuclear localization sequence (NLS) was added to the α-syn coding sequence. Virus-mediated gene transfer, behavioural tests, RNA-Seq, immunohistochemistry, western blotting, and quantitative real-time PCR were then performed.ResultsIn vivo experiments using a mouse model showed that nuclear α-syn increased the severity of the PD-like phenotype, including the loss of dopaminergic neurons concomitant with motor impairment and the formation of α-syn inclusions. These nuclear inclusions contained α-syn species of high molecular weights and induced strong transcriptional dysregulation, especially induced high expression of p21 and senescence-associated secretory phenotype (SASP)-related genes. In addition, the transcriptional alterations induced by nuclear α-syn were associated with gliosis, inflammation, oxidative and DNA damage, and lysosomal dysfunction, and they eventually accelerated neuronal loss and neurodegeneration.ConclusionsOur results suggest that nuclear α-syn plays a crucial role in PD pathogenesis.

An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma.

Cancer cells have remarkable plasticity allowing them to acquire many biological states. Melanoma cells have the ability to switch from a proliferative melanocytic state to an invasive mesenchymal state and back again resulting in intratumoral heterogeneity. While microphthalmia-associated transcription factor (MITF) promotes the melanocytic phenotype, it is unclear what transcription factors drive the mesenchymal phenotype, and what mechanisms regulate the switch from the proliferative state to the mesenchymal state. We show that nuclear localization of the MITF paralog TFE3 correlates positively with metastatic potential in melanoma cell lines and tumors, and that deletion of TFE3 in MITF-low melanoma cell lines eliminates migration and metastatic ability. Further, we find that MITF suppresses the mesenchymal phenotype by activating expression of FNIP2, which encodes a component of an mTORC1-stimulated pathway promoting cytoplasmic retention and lysosomal degradation of TFE3. These findings point to the mTOR pathway and TFE3 as key regulators of melanoma plasticity.

Loss of Lamin A leads to the nuclear translocation of AGO2 and compromised RNA interference.

In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event; however, in the last decade, a growing number of reports convincingly show the nuclear localization of the Argonaute (AGO) proteins. Nevertheless, the extent of nuclear RNAi and its implication in biological mechanisms remain to be elucidated. We found that reduced Lamin A levels significantly induce nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 cells, evident by changes in cell morphology, increased cell proliferation, and oncogenic miRNA expression. Moreover, AGO fPAR-CLIP in Lamin A KO SHSY5Y cells revealed significantly reduced RNAi activity. Further exploration of the nuclear AGO interactome by mass spectrometry identified FAM120A, an RNA-binding protein and known interactor of AGO2. Subsequent FAM120A fPAR-CLIP, revealed that FAM120A co-binds AGO targets and that this competition reduces the RNAi activity. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, FAM120A mediated RNAi impairment, and upregulation of oncogenic miRNAs, facilitating cancer cell proliferation.

The interaction between the import carrier Hikeshi and HSP70 is modulated by heat, facilitating the nuclear import of HSP70 under heat stress conditions.

Heat stress strongly triggers the nuclear localization of the molecular chaperone HSP70. Hikeshi functions as a unique nuclear import carrier of HSP70. However, how the nuclear import of HSP70 is activated in response to heat stress remains unclear. Here, we investigated the effects of heat on the nuclear import of HSP70. In vitro transport assays revealed that pretreatment of the test samples with heat facilitated the nuclear import of HSP70. Furthermore, binding of Hikeshi to HSP70 increased when temperatures rose. These results indicated that heat is one of the factors that activates the nuclear import of HSP70. Previous studies showed that the F97A mutation in Hikeshi in an extended loop induced an opening in the hydrophobic pocket and facilitated the translocation of Hikeshi through the nuclear pore complex. We found that nuclear accumulation of HSP70 occurred at a lower temperature in cells expressing the Hikeshi-F97A mutant than in cells expressing wild-type Hikeshi. Collectively, our results show that the movement of the extended loop may play an important role in the interaction of Hikeshi with both FG (phenylalanine-glycine)-nucleoporins and HSP70 in a temperature-dependent manner, resulting in the activation of nuclear import of HSP70 in response to heat stress.

Atypical structure of the nuclear membrane, distribution of nuclear pores and lamin B1 in spermatozoa of patients with complete and partial globozoospermia.

Globozoospermia is a form of male infertility characterized by spermatozoa with spherical heads lacking acrosomes. The aim of this study was to evaluate ultrastructural and molecular defects in different types of globozoospermia. Semen samples from 12 infertile patients (9 with complete globozoospermia and 3 with partial globozoospermia) and 10 normozoospermic men (control) were examined by transmission electron microscopy and immunocytochemistry with antibodies against lamin B1. The presence of lamin A and progerin was assessed by reverse transcription-PCR. Whole exome sequencing was performed in three patients. In semen samples with complete and partial globozoospermia, lamin B1 was observed at the periphery of sperm nuclei, whereas lamin A and progerin were absent. Nuclear envelope pores were found in spermatozoa from both patient groups, regardless of morphology and chromatin condensation, in contrast to the control group. Non-condensed chromatin was present in 51%-81% of cases of complete globozoospermia and in 36%-79% of cases of partial globozoospermia. Homozygous DPY19L2 and SPATA16 variants were identified in two patients with partial globozoospermia and one patient with complete globozoospermia. An atypical nuclear membrane with abnormal nuclear pore distribution and lamin B1 localization was observed in spermatozoa from patients with both complete and partial globozoospermia. The genetic defects in the DPY19L2 and SPATA16 genes detected in patients from both globozoospermic groups suggest a generalized disruption of nuclear structure in globozoospermia, highlighting the genetic and phenotypic similarities between complete and partial globozoospermia.

The marine-derived HIF-1α inhibitor, Yardenone 2, reduces prostate cancer cell proliferation by targeting HIF-1 target genes

BackgroundProstate cancer (PCa) ranks as the second most prevalent cancer in men, with advanced stages posing significant treatment challenges. Given its solid tumor nature, PCa is highly susceptible to hypoxia, a condition associated with resistance to radiation and chemotherapy, metastasis, and unfavorable patient outcomes. Hypoxia-inducible factors (HIFs) play a pivotal role in cancer cell adaptation to hypoxic environments, contributing to treatment resistance. Consequently, inhibitors targeting HIFs hold promise for cancer therapy.MethodsIn this study, we aimed to characterize novel HIF-1α inhibitors including Sodwanones A (1), B (2), C (3), G (4) and Yardenone 2 (5) isolated from marine sponges belonging to the Axinella genus. Our investigation evaluated the impact of these compounds on various aspects of HIF-1α regulation, including stabilization, nuclear localization, expression of HIF-1 target genes (while sparing HIF-2 target genes), cellular metabolism, as well as cell proliferation and viability in prostate cells under hypoxic conditions.ResultsOur findings revealed that among the compounds tested, Yardenone 2 exhibited notable effects in hypoxia: it destabilized HIF-1α at the protein level, decreased its nuclear localization, selectively altered the expression of HIF-1 target genes, and restrained cell proliferation in aggressive PC3 prostate cancer cells as well as in an MSK-PCa3 patient-derived organoid line. Moreover, it affected the morphology of these organoid. Yardenone 2 was also compared to Docetaxel, a specific microtubule inhibitor and a drug used in the treatment of prostate cancer. The comparison between the two compounds revealed notable differences, such as a lack of specificity to hypoxic cells of Docetaxel.ConclusionThese results mark the first demonstration that Yardenone 2 functions as a cytostatic-like inhibitor impacting microtubules, specifically targeting hypoxic cancer cells. This discovery suggests a promising avenue for novel therapeutic interventions in prostate cancer.

Identification of new benzofuran derivatives as STING agonists with broad-spectrum antiviral activity

The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-β promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-β transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the μM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN-dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING-dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals.