Nuclear Localization Research Articles

Bioavailable testosterone and androgen receptor activation, but not total testosterone, are associated with muscle mass and strength in females.

Testosterone, the major androgen, influences the reproductive and non-reproductive systems in males and females via binding to the androgen receptor (AR). Both circulating endogenous testosterone and muscle AR protein content are positively associated with muscle mass and strength in males, but there is no such evidence in females. Here, we tested whether circulating testosterone levels were associated with muscle mass, function, or the muscle anabolic response to resistance training in pre-menopausal females. Twenty-seven pre-menopausal, untrained females (aged 23.5 ± 4.8 years) underwent a 12-week resistance training programme. Muscle strength, size, power, and plasma and urine androgen hormone levels were measured. Skeletal muscle biopsies were collected before and after the training programme to quantify the effect of resistance training on AR content and nuclear localisation. Primary muscle cell lines were cultured from a subset (n=6) of the participants' biopsies and treated with testosterone to investigate its effect on myotube diameter, markers of muscle protein synthesis and AR cellular localisation. Physiological levels of total testosterone were not associated with muscle mass or strength at baseline or with the changes in muscle mass and strength that occurred in response to resistance training in our cohort of pre-menopausal females. In contrast, bioavailable testosterone and the proportion of nuclear-localised AR were positively associated with skeletal muscle mass and strength in pre-menopausal females. In vitro, supra-physiological doses of testosterone increased myocyte diameter, but this did not occur via the Akt/mTOR pathway as previously suggested. Instead, we show a marked increase in AR nuclear localisation with testosterone administration in vitro. KEY POINTS: Total circulating testosterone was not related to muscle mass or strength before or after resistance training in pre-menopausal females. Bioavailable testosterone was positively related to exercise-induced muscle hypertrophy in pre-menopausal females. In vivo nuclear localisation of the androgen receptor was positively related to muscle mass in pre-menopausal females at baseline, but not to resistance training-induced hypertrophy. Testosterone treatment induced androgen receptor nuclear translocation but did not induce mTOR signalling in primary skeletal myocytes cultured from pre-menopausal female muscle.

PAM-relaxed and temperature-tolerant CRISPR-Mb3Cas12a single transcript unit systems for efficient singular and multiplexed genome editing in rice, maize, and tomato.

Class 2 Type V-A CRISPR-Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T-rich genomic regions, complementing the widely used CRISPR-Cas9 in plants. To enhance the utility of Cas12a, we investigate three Cas12a orthologs-Mb3Cas12a, PrCas12a, and HkCas12a-in plants. Protospacer adjacent motif (PAM) requirements, editing efficiencies, and editing profiles are compared in rice. Among these orthologs, Mb3Cas12a exhibits high editing efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than the canonical TTTV PAM of LbCas12a and AsCas12a. To optimize Mb3Cas12a, we develop an efficient single transcription unit (STU) system by refining the linker between Mb3Cas12a and CRISPR RNA (crRNA), nuclear localization signal (NLS), and direct repeat (DR). This optimized system enables precise genome editing in rice, particularly for fine-tuning target gene expression by editing promoter regions. Further, we introduced Arginine (R) substitutions at Aspartic acid (D) 172, Asparagine (N) 573, and Lysine (K) 579 of Mb3Cas12a, creating two temperature-tolerant variants: Mb3Cas12a-R (D172R) and Mb3Cas12a-RRR (D172R/N573R/K579R). These variants demonstrate significantly improved editing efficiency at lower temperatures (22 °C and 28 °C) in rice cells, with Mb3Cas12a-RRR showing the best performance. We extend this approach by developing efficient Mb3Cas12a-RRR STU systems in maize and tomato, achieving biallelic mutants targeting single or multiple genes in T0 lines cultivated at 28 °C and 25 °C, respectively. This study significantly expands Cas12a's targeting capabilities in plant genome editing, providing valuable tools for future research and practical applications.

The highly conserved region within exonuclease III-like in PML-I regulates the cytoplasmic localization of PML-NBs

The sub-nuclear protein structure PML-NB regulates a wide range of important cellular functions, while its abnormal cytoplasmic localization may have pathological consequences. However, the nature of this aberrant localization remains poorly understood. In this study, we unveil that PML-I, the most conserved and abundant structural protein of PML-NB, possesses potent cytoplasmic targeting ability within the N-terminal half of the exonuclease III-like domain encoded by its unique exon 9, independent of the known nuclear localization signal. Fusion of this region to PML-VI can relocate PML-VI from the nucleus to the cytosol. Structural and deletion analysis revealed that the cytoplasmic targeting ability of this domain was restrained by the sequences encoded by exon 8a and the 3’ portion of exon 9 in PML-I. Deletion of either of these regions relocates PML-I to the cytosol. Furthermore, we observed a potential interaction between the ER-localized TREX1 and the cytoplasmic-located PML-I mutants. Our results suggest that perturbation of the EXO-like domain of PML-I may represent an important mode to translocate PMLs from the nucleus to the cytosol, thereby interfering with the normal nuclear functions of PML-NBs.

Wedelolactone alleviates inflammation and cartilage degeneration by suppressing the NF-κB signaling pathway in osteoarthritis

Inflammation and extracellular matrix (ECM) degradation are two major factors involved in the pathogenesis of osteoarthritis (OA). Wedelolactone, a natural compound classified as a coumestan, is isolated from the medicinal plants Eclipta alba and Wedelia calendulacea. In this study, we assessed the protective effects of Wedelolactone on chondrocytes in OA. Our findings show that pretreatment with Wedelolactone effectively inhibited the IL-1β-induced upregulation of COX‑2, iNOS, TNF-α, and IL6 in chondrocytes, contributing to inflammation suppression. Moreover, pretreatment with Wedelolactone followed by IL-1β treatment significantly increased the expression of Collagen II and SOX9, while decreasing the expression of Adamts5, MMP1, MMP3, and MMP13, thereby promoting ECM protection. Through Network pharmacology Analysis, we identified 14 key targets that link Wedelolactone and OA. GO and KEGG pathway analysis suggested that Wedelolactone primarily impacted OA by targeting inflammatory responses, particularly the NF-κB signaling pathway. Further studies demonstrated Wedelolactone prevented IL-1β-induced activation of NF-κB signaling pathway by inhibiting the translocation of p65 and the preventing the degradation of IκBα in human chondrocytes. Molecular docking studies also indicated that Wedelolactone can directly bind to the NF-κB complex, thereby inhibited the nuclear localization of p65. In vivo experiments demonstrated that Wedelolactone can alleviate cartilage damage in DMM mice model. In summary, Wedelolactone appears to mitigate inflammation and cartilage degeneration by suppressing the NF-κB signaling pathway, thereby alleviating OA progression. Our results suggested Wedelolactone may offer therapeutic advantages for OA treatment.

KLF4 regulates trophoblast function and associates with unexplained recurrent spontaneous abortion

BackgroundRecurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells.MethodsWe firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration.ResultsKLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo.ConclusionsThe KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function.

SARS-CoV-2 Nucleocapsid Protein Antagonizes GADD34-Mediated Innate Immune Pathway through Atypical Foci.

The integrated stress response, especially stress granules (SGs), contributes to host immunity. Typical G3BP1+ stress granules (tSGs) are usually formed after virus infection to restrain viral replication and stimulate innate immunity. Recently, several SG-like foci or atypical SGs (aSGs) with proviral function have been found during viral infection. We have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein induces atypical N+/G3BP1+ foci (N+foci), leading to the inhibition of host immunity and facilitation of viral infection. However, the precise mechanism has not been well clarified yet. In this study, we showed that the SARS-CoV-2 N (SARS2-N) protein inhibits dsRNA-induced growth arrest and DNA damage-inducible 34 (GADD34) expression. Mechanistically, the SARS2-N protein promotes the interaction between GADD34 mRNA and G3BP1, sequestering GADD34 mRNA into the N+foci. Importantly, we found that GADD34 participates in IRF3 nuclear translocation through its KVRF motif and promotes the transcription of downstream interferon genes. The suppression of GADD34 expression by the SARS2-N protein impairs the nuclear localization of IRF3 and compromises the host's innate immune response, which facilitates viral replication. Taking these findings together, our study revealed a novel mechanism by which the SARS2-N protein antagonized the GADD34-mediated innate immune pathway via induction of N+foci. We think this is a critical strategy for viral pathogenesis and has potential therapeutic implications.

GL-V9 Promotes Autophagy-Mediated YAP1 Degradation and Activates Mitochondrial Apoptosis in PDAC Cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive forms of pancreatic cancer with a poor prognosis. YAP1 expression is markedly elevated in PDAC, but how it works is not clear. GL-V9, a derivative of the natural compound wogonin, effectively fights a variety of tumors; however, its effect on PDAC has not yet been studied. Methods: TCGA database analysis, Western blots, immunofluorescence, and real-time PCR were used to evaluate GL-V9's effect on YAP1 expression and mRNA levels. Immunofluorescence was used to examine the co-location of YAP1 with LAMP2 and p62. Co-immunoprecipitation was used to assess the binding of YAP1 to ubiquitin, p62, and TEAD1. A PDAC graft tumor model was used to test GL-V9's pharmacological effects. Western blots and immunohistochemistry were used to measure apoptosis- and autophagy-related protein expression. Results: GL-V9 effectively promoted the degradation of YAP1, reduced YAP1 nuclear localization, and induced mitochondrial apoptosis in PDAC cells. YAP1 overexpression led to the upregulation of Bcl-2 and attenuated the caspase cascade induced by GL-V9. Furthermore, we demonstrated that GL-V9 induced autophagosome-lysosome fusion via the AKT/mTOR/TFEB pathway, leading to mitochondrial apoptosis in PDAC cells. In vivo studies also confirmed that GL-V9 exerts anti-tumor effects by suppressing YAP1 expression, while also activating autophagy and inducing mitochondrial apoptosis in BXPC-3-bearing BALB/c nude mice. Conclusions: Our findings underscore the importance of autophagy-mediated YAP1 degradation in PDAC, providing a novel molecular rationale (GL-V9) as a promising treatment for this disease.

Nuclear localization of APLF facilitates breast cancer metastasis

Most breast cancer deaths result from metastases. We previously reported that DNA repair factor and histone chaperone Aprataxin PNK-like Factor (APLF) is involved in EMT-associated metastasis of triple negative breast cancer (TNBC) cells. However, non-metastatic cells also expressed APLF, the implications of which in disease advancement remain uncertain. Here, we demonstrate that the metastatic prognosis of breast cancer cells may be determined by the cellular localization of APLF. Using TNBC patient samples and cell lines, we discovered that APLF was localized in the nucleus and cytoplasm, whereas other subtypes of breast cancer had cytosolic or perinuclear localization. To investigate metastatic properties in vitro and in vivo, we modeled APLF differential localization by stably producing APLF-tagged nuclear localization signal (NLS) in the luminal subtype MCF7 cells in the absence of putative APLF NLS. Nuclear APLF in non-metastatic MCF7 cells demonstrated pronounced migration, invasion and metastatic potential. We obtained the mechanistic insight from molecular studies that PARP1 could facilitate the transport of APLF from the cytosol to the nucleus, assisting in the metastasis of TNBC cells linked with EMT. Inhibition of PARP1 enzymatic activity with olaparib abrogated the nuclear expression of APLF with loss in expression of genes associated with EMT. Thus, our findings reveal that cellular localization of APLF may predict the risk of breast cancer to metastasize and hence could be exploited to determine the disease progression. We anticipate that the inhibition of cytosolic PARP1-APLF interaction may potentially aid in the prevention of breast cancer metastasis in TNBC patients.

The mutated in colorectal cancer (MCC) gene can serve as a potential biomarker of glioblastoma.

The mutated in colorectal cancer (MCC) gene was initially identified as a candidate tumor suppressor gene in colorectal cancer, acting as a negative regulator of cell cycle progression. However, its functional roles in brain tumors, particularly glioblastoma, remain largely unexplored. This study reveals a significant association between MCC status and glioblastoma. We explored MCC expression in the glioblastoma database, patient samples, and cell lines. We investigated the proliferation and migration of the cell lines in MCC gene knockdown using small interfering RNA. In vitro analyses revealed elevated protein and mRNA levels of MCC in several glioblastoma cell lines (U118MG and T98G). Silencing MCC expression via siRNA-mediated knockdown resulted in increased proliferation and migration of these cell lines. Supporting these findings, analyses of The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and Genotype-Tissue Expression (GTEx) databases confirmed higher MCC expression in glioblastoma tumors than in normal brain tissue. Importantly, we observed that high MCC expression was associated with poor prognosis in glioblastoma patients, highlighting its potential role in disease progression. Additionally, this study identifies a nuclear localization of MCC in the glioblastoma cell line. These findings indicate that MCC expression is significantly upregulated in glioblastoma and may play a role in its pathophysiology, warranting further investigation.

8028 Targeted Therapy-Induced ER And HER2 Chromatin Binding: A Novel Driver Of Poor Response To Treatment In Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

8028 Targeted Therapy-Induced ER and HER2 Chromatin Binding: a Novel Driver of Poor Response to Treatment in Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

7157 Unveiling PROP1 protein complex: NFIB as a key candidate in pituitary development

Abstract Disclosure: L.C. Iglesias García: None. M.F. Mercogliano: None. C. Schuster: None. L. Cheung: None. F. Brunello: None. V. Michan: None. M. Waldman: None. L. Defelipe: None. M. Martí: None. S.A. Camper: None. M.I. Perez-Millan: None. The transcription factor PROP1 is essential for pituitary development, and mutations in the gene are the most common cause of hypopituitarism in children. To understand PROP1 function, it is essential to identify the protein components of the PROP1 transcription complex. Rapid immunoprecipitation and mass spectrometry (RIME) experiments in GHFT1 cells, genetically engineered to express biotin-tagged PROP1, have revealed the association of PROP1 with 43 transcription factors (including β-CATENIN and the Nuclear Factor I/B). In silico analysis showed association between the armadillo domains of β-CATENIN and a predicted nuclear localization signal motif of PROP1. This interaction between PROP1 and β-CATENIN was confirmed in pituitary GHFT1 cells, where over-expression of PROP1 induces the translocation of β-CATENIN from the membrane and cytoplasm to the nucleus. Moreover, in pituitaries from Prop1df/df mutant embryos at e12.5, β-CATENIN was predominantly membrane-bound compared to wild-type embryos. These findings suggest that PROP1 is necessary for translocation of β-CATENIN to the nucleus during pituitary development.The association of PROP1 with NFIB is intriguing because PROP1 is necessary for the epithelial to mesenchymal-like transition of stem cells in pituitary development, and NFIB is associated with induction of EMT in several cancer models. We used immunohistochemistry to show that NFIB is expressed in embryonic pituitary glands from e12.5 to e16.5, and it co-localizes with the stem cell marker SOX2. To determine whether PROP1 and NFIB have common downstream targets, we performed CUT&RUN experiments for NFIB in GHFT-1 cells and compared the results with those from PROP1 ChIP-seq. Both PROP1 and NFIB bind to three sites in the NFIB gene, suggesting that NFIB is a downstream target of PROP1 and subject to autoregulation. Consistent with this idea, NFIB is reduced in pituitaries from Prop1df/df embryos at both e12.5 and e14.5 relative to normal littermates. Integration of the results from PROP1 ChIP-seq and RNA-seq (GHFT1 vs PROP1-GHFT1 cells) with NFIB CUT&RUN generated a list of 190 potential target genes. These genes are associated with cell migration, emphasizing their correlation with the EMT-like process orchestrated by PROP1 during pituitary development. In sum, we have identified an interesting set of PROP1 interacting proteins, including the transcription factor NFIB, which plays an important role in regulating stem cell differentiation in several organ systems. We have also cataloged the genome-wide set of binding sites for PROP1 and NFIB, which overlap at many genes regulating cell migration, a process that is important for pituitary stem cells to leave the niche and initiate differentiation. Presentation: 6/3/2024

B-328 A Novel Pathway of Akt-Dependent PARP1 Activation Through the Nuclear Localization of EPRS1

Abstract Background Glutamyl-prolyl-tRNA synthetase (EPRS1) is the only bifunctional aminoacyl-tRNA synthetase (aaRS) and is essential for interpreting the genetic code. EPRS1, along with seven other aaRSs, forms the multi-tRNA synthetase complex (MSC). EPRS1 consists of two catalytic domains, GluRS and ProRS, connected by a linker region. Several aaRSs within the MSC, including EPRS1, can dissociate from the MSC in response to specific stimuli. This dissociation enables non-canonical activities distinct from their primary role in protein synthesis. Breast cancer cells harboring a loss-of-function mutation in phosphatase and tensin homolog (PTEN) are known to have more activity in the serine/threonine kinase Akt. Poly-(ADP)-ribose polymerase 1 (PARP1), plays a vital role in DNA damage repair (DDR). PARP1 deposits one or more ADP-Ribose moieties onto damaged DNA or other proteins in a process referred to as PARylation. We hope to connect Akt activation and PARP1 activity through the nuclear localization and noncanonical function of EPRS1. We hypothesize, that when Akt is activated, either through the loss of PTEN function or through stimuli such as heat shock or H2O2 treatment, will stimulate the nuclear localization of EPRS1 and modulate PARP1 activity. Methods To test our hypothesis, we evaluate Akt activation, EPRS1 localization, and PARP1 activity. Using pharmacological activators and inhibitors of Akt, we test if inhibitors and activators alone are sufficient to cause the nuclear localization of EPRS1. Biochemically, we isolate nuclear and cytosolic fractions used in various western blot analysis. We use a variety of assays for PARP1 activity and DDR that include immobilized histones, calculating IC50s of PARP1 inhibitors, comet assay, and confocal microscopy. To ascertain the connection between PARP1 and EPRS1 we use LC-MS/MS analysis and immunoprecipitation. Results We show, in both patient-derived and cultured breast cancer cells, elevated levels of EPRS1 not only in the cytoplasm, but also within the nucleus. We find that nuclear localization EPRS1 is facilitated by a nuclear localization sequence (NLS) located within the linker region of the protein. Breast cancer cells harboring a loss-of-function in PTEN exhibit higher levels of EPRS1 in the nucleus compared to PTEN-positive cells. In EPRS1 knockdown cells, there is diminished expression of multiple tumor-related transcription factors, DNA-damage repair, tumor cell survival, and PARP1 auto-PARylation. We demonstrate that nuclear EPRS1 binds to and shares an interactome with PARP1. Conclusions EPRS1-mediated regulation of PARP1 activity provides a new mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the non-canonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, might provide a new therapeutic approach in breast and other forms of cancer, potentially supplementing existing cancer therapeutics.

A new class of capsid-targeting inhibitors that specifically block HIV-1 nuclear import.

HIV-1 capsids cross nuclear pore complexes (NPCs) by engaging with the nuclear import machinery. To identify compounds that inhibit HIV-1 nuclear import, we screened drugs in silico on a three-dimensional model of a CA hexamer bound by Transportin-1 (TRN-1). Among hits, compound H27 inhibited HIV-1 with a low micromolar IC50. Unlike other CA-targeting compounds, H27 did not alter CA assembly or disassembly, inhibited nuclear import specifically, and retained antiviral activity against PF74- and Lenacapavir-resistant mutants. The differential sensitivity of divergent primate lentiviral capsids, capsid stability and H27 escape mutants, together with structural analyses, suggest that H27 makes multiple low affinity contacts with assembled capsid. Interaction experiments indicate that H27 may act by preventing CA from engaging with components of the NPC machinery such as TRN-1. H27 exhibited good metabolic stability in vivo and was efficient against different subtypes and circulating recombinant forms from treatment-naïve patients as well as strains resistant to the four main classes of antiretroviral drugs. This work identifies compounds that demonstrate a novel mechanism of action by specifically blocking HIV-1 nuclear import.

Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals

Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in in vitro situations differed from that in in vivo.

Nup210 Promotes Colorectal Cancer Progression by Regulating Nuclear Plasma Transport

The nuclear pore complex (NPC) regulates nucleoplasmic transport, transcription, and genomic integrity in eukaryotic cells. However, little is known about how NPC works in cancer. In this study, we investigated the role of the nuclear pore protein 210 (Nucleoporin 210, Nup210) in colorectal cancer (CRC). Bioinformatics analysis revealed that the expression of Nup210 was increased in CRC and was associated with poor patient prognosis, but it was not a statistically significant independent prognostic factor. Moreover, knockdown of Nup210 in CRC cells inhibited the proliferation, invasion, and metastasis of CRC cells in vivo and in vitro. Additionally, nuclear size and nuclear plasma material transport capacity decreased along with the number and density of NPCs on the surface of CRC cells when Nup210 expression was inhibited. Furthermore, Nup210 required nuclear localization sequences (NLS) to localize to the nuclear membrane surface and interact with importin-α/β, which in turn affected the transit of nuclear plasma material. Importazole, a small molecule inhibitor of importin, along with therapy that targets the Nup210 protein is anticipated to be a novel strategy for CRC treatment. Their combination may be able to more effectively lower CRC tumor load. In conclusion, Nup210 modulates cellular nucleoplasmic transport capability and cell surface NPC density via NLS, thus promoting CRC progression. This discovery validates the molecular function of NPC in the development of CRC and provides a theoretical foundation for NPC-regulated nuclear import targeting as a therapeutic strategy for CRC.

Squash vein yellowing virus from California emerged in the Middle East via intragenic and intergeneric recombination events in the hypervariable potyvirus P1 and ipomovirus P1a genes.

We present the complete sequence of the genomic RNA of an isolate of squash vein yellowing virus (Ipomovirus cucurbitavenaflavi) from California (SqVYV-CA) and show it is a recombinant virus with a highly divergent 5' UTR and proximal P1a gene. The evolution of SqVYV-CA involved an intrageneric event between unknown potyviruses, related to isolates of papaya ringspot virus (Potyvirus papayanuli) from the Old World, and an intergeneric event between this recombinant potyvirus (minor parent) and an isolate of SqVYV from Israel (SqVYV-IL) (major parent). These events occurred in mixed infections and in the potyvirus P1 and ipomovirus P1a recombination hotspots and resulted in SqVYV-CA having a potyvirus 5' UTR and chimeric P1-P1a gene/protein and the remainder of the genome from SqVYV-IL. The SqVYV-CA chimeric P1-P1a gene is under positive selection, and the protein is intrinsically disordered and may localize to the nucleus via nuclear localization signals in the P1 part. The C-terminal SqVYV-IL P1a part also diverged but retained the conserved serine protease motif. Furthermore, substantial divergence in SqVYV isolates from the Middle East was associated with genetic drift and a long evolutionary history in this region. The finding that the host range and symptomatology in cucurbits of SqVYV-CA is similar to those of SqVYV from Florida and SqVYV-IL, indicated that the recombinant part of the genome had no obvious effect on the virus-host interaction. A divergent part of the P1 sequence of the SqVYV-CA P1-P1a gene was used to develop a primer pair and RT-PCR test for specific detection of SqVYV-CA. This test was used to detect spread of SqVYV-CA to a new production area of California in 2021 and 2022. Together, these results demonstrate (i) a high level of genetic diversity exists among isolates of SqVYV and involved intra- and intergeneric recombination and genetic drift (mutation), (ii) evidence that SqVYV originated in the Middle East and that there were independent introductions into the New World and (iii) the remarkable genetic flexibility of the 5' proximal genes of these viruses.

Circular RNA hsa_circ_0081343 modulates trophoblast autophagy through Rbm8a nuclear translocation

IntroductionFetal growth restriction (FGR) is a kind of obstetric complication that seriously endangers fetal life. Recent studies reported significant reduction of hsa_circ_0081343 in human placenta developed in FGR and is involved in cell migration, invasion, and apoptosis of trophoblast by acting as microRNA sponges. Autophagy is required for invasion of trophoblast cells and for vascular remodeling during placentation. In this study, we aimed to explore the mechanistic link between hsa_circ_0081343 and autophagy. MethodsWe investigated the interactions between hsa_circ_0081343 and RNA-binding proteins were studied by RNA pull-down assay, mass spectrometry and RNA immunoprecipitation assay. The mechanism of nuclear translocation of Rbm8a were assessed by reverse transcription-quantitative PCR, Western blot, immunofluorescence and Co-Immunoprecipitation. Western blot, immunofluorescence and transmission electron microscopy were performed to elucidate the mechanism underlying hsa_circ_0081343 and/or Rbm8a mediated regulation of autophagy. Resultshsa_circ_0081343 served as an RNA-binding protein (RBP) sponge. RNA binding motif protein 8A (Rbm8a) was directly bound to hsa_circ_0081343 in the cytoplasm, while knockdown of hsa_circ_0081343 facilitated Rbm8a localization in the nucleus. We also identified Rbm8a as a potential import cargo for Importin13 (Ipo13), which transported Rbm8a across the nuclear membrane into the nucleus.Ipo13 recognized Rbm8a via a functional nuclear localization signal (NLS). Furthermore, the mechanistic study revealed that hsa_circ_0081343-mediated nuclear translocation of Rbm8a activated trophoblast autophagy. DiscussionOur results suggest that hsa_circ_0081343 could bind to RBP and the interaction between hsa_circ_0081343 and Rbm8a participate in regulating autophagy. These findings offer novel molecular targets and insights for a potential therapeutic strategy against FGR.

Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.

HSPA12A stimulates "Smurf1-Hif1α-aerobic glycolysis" axis to promote proliferation of renal tubular epithelial cells after hypoxia/reoxygenation injury.

Proliferation of renal tubular epithelial cells (TECs) is critical for the recovery after kidney ischemia/reperfusion (KI/R). However, there is still a lack of ideal therapies for promoting TEC proliferation. Heat shock protein A12A (HSPA12A) shows abundant expression in kidney in our previous studies. To investigate the role of HSPA12A in TEC proliferation after KI/R, an in vitro KI/R model was simulated by hypoxia (12h) and reoxygenation (12h) in human kidney tubular epithelial HK-2 cells. We found that, when hypoxia/reoxygenation (H/R) triggered HK-2 cell injury, HSPA12A expression was downregulated, and extracellular lactate, the readout of glycolysis, was also decreased. Loss and gain of functional studies showed that HSPA12A did not change cell viability after hypoxia but increased cell proliferation as well as glycolytic flux of HK-2 cells after H/R. When blocking glycolysis by 2-deoxy-D-glucose or oxamate, the HSPA12A promoted HK-2 cell proliferation was also abolished. Further analysis revealed that HSPA12A overexpression increased hypoxia-inducible factor 1α (Hif1α) protein expression and nuclear localization in HK-2 cells in response to H/R, whereas HSPA12A knockdown showed the opposite effects. Notably, pharmacologicalinhibition of Hif1α with YC-1 reversed the HSPA12A-induced increases of both glycolytic flux and proliferation of H/R HK-2 cells. Moreover, the HSPA12A increased Hif1α protein expression was not via upregulating its transcription but through increasing its protein stability in a Smurf1-dependent manner. The findings indicate that HSPA12A might serve as a promising target for TEC proliferation to help recovery after KI/R.