Nuclear Protein Research Articles

The actin-binding protein palladin associates with the respiratory syncytial virus matrix protein.

The respiratory syncytial virus (RSV) matrix (M) protein plays an important role in infection as it can interact with viral components as well as the host cell actin microfilaments. The M-actin interaction may play a role in facilitating the transportation of virion components to the apical surface, where RSV is released. We show that M protein's association with actin is facilitated by palladin, an actin-binding protein. Cells were infected with RSV or transfected to express full-length M as a green fluorescent protein (GFP)-tagged protein, followed by removal of nuclear and cytosolic proteins to enrich for cytoskeleton and its associated proteins. M protein was present in inclusion bodies tethered to microfilaments in infected cells. In transfected cells, GFP-M was presented close to microfilaments, without association, suggesting the possible involvement of an additional protein in this interaction. As palladin can bind to proteins that also bind actin, we investigated its interaction with M. Cells were co-transfected to express GFP-M and palladin as an mCherry fluorescent-tagged protein, followed by cytoskeleton enrichment. M and palladin were observed to colocalize towards microfilaments, suggesting that palladin is involved in the M-actin interaction. In co-immunoprecipitation studies, M was found to associate with two isoforms of palladin, of 140 and 37 kDa. Interestingly, siRNA downregulation of palladin resulted in reduced titer of released RSV, while cell associated RSV titer increased, suggesting a role for palladin in virus release. Together, our data show that the M-actin interaction mediated by palladin is important for RSV budding and release.IMPORTANCERespiratory syncytial virus is responsible for severe lower respiratory tract infections in young children under 5 years old, the elderly, and the immunosuppressed. The interaction of the respiratory syncytial virus matrix protein with the host actin cytoskeleton is important in infection but has not been investigated in depth. In this study, we show that the respiratory syncytial virus matrix protein associates with actin microfilaments and the actin-binding protein palladin, suggesting a role for palladin in respiratory syncytial virus release. This study provides new insight into the role of the actin cytoskeleton in respiratory syncytial virus infection, a key host-RSV interaction in assembly. Understanding the mechanism by which the RSV M protein and actin interact will ultimately provide a basis for the development of therapeutics targeted at RSV infections.

Quercetin ameliorates senescence and promotes osteogenesis of BMSCs by suppressing the repetitive element‑triggered RNA sensing pathway.

Cell senescence impedes the self‑renewal and osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs), thus limiting their application in tissue regeneration. The present study aimed to elucidate the role and mechanism of repetitive element (RE) activation in BMSC senescence and osteogenesis, as well as the intervention effect of quercetin. In an H2O2‑induced BMSC senescence model, quercetin treatment alleviated senescence as shown by a decrease in senescence‑associated β‑galactosidase (SA‑β‑gal)‑positive cell ratio, increased colony formation ability and decreased mRNA expression of p21 and senescence‑associated secretory phenotype genes. DNA damage response marker γ‑H2AX increased in senescent BMSCs, while expression of epigenetic markers methylation histone H3 Lys9, heterochromatin protein 1α and heterochromatin‑related nuclear membrane protein lamina‑associated polypeptide 2 decreased. Quercetin rescued these alterations, indicating its ability to ameliorate senescence by stabilizing heterochromatin structure where REs are primarily suppressed. Transcriptional activation of REs accompanied by accumulation of cytoplasmic double‑stranded (ds)RNA, as well as triggering of the RNA sensor retinoic acid‑inducible gene I (RIG‑I) receptor pathway in H2O2‑induced senescent BMSCs were shown. Similarly, quercetin treatment inhibited these responses. Additionally, RIG‑I knockdown led to a decreased number of SA‑β‑gal‑positive cells, confirming its functional impact on senescence. Induction of senescence or administration of dsRNA analogue significantly hindered the osteogenic capacity of BMSCs, while quercetin treatment or RIG‑I knockdown reversed the decline in osteogenic function. The findings of the current study demonstrated that quercetin inhibited the activation of REs and the RIG‑I RNA sensing pathway via epigenetic regulation, thereby alleviating the senescence of BMSCs and promoting osteogenesis.

Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants.

O-fucose, a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis, Toxoplasma gondii , and an unrelated protist, the social amoeba Dictyostelium discoideum . Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected using Aleuria aurantia lectin (AAL), but in most organisms AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such as Acanthamoeba castellanii .

Homeostatic nanomedicine: Protein S-nitrosylation dyshomeostasis dominated oncotherapy based on a tumor-microenvironment-activated nanoparticle

Nitric oxide (NO) holds promise as a therapeutic agent in oncotherapy, yet its efficacy is hindered by low bioavailability, concentration-dependency, and tumor resistance to nitrosative stress. To overcome these limitations, herein, we propose a novel strategy aimed at specifically breaking the protein S-nitrosylation homeostasis of tumors. An acid-responsive nanoplatform comprising amorphous iron / mesoporous silica (AMSP) is constructed to orchestrate synergistic anti-tumor effect of Fe2+ and NO. Activating by the tumor microenvironment, nanopores of AMSP facilitate the in-situ generation of dinitrosyl iron complexes, promoting the physiological stability of NO and protein S-nitrosylation via transnitrosylation. Moreover, the released Fe2+ accelerate reactive oxygen species accumulation in tumor cells, which not only derives stronger nitrating reagents, but also inhibits the denitrosylation reaction triggered by tumor-expressed reducing species. The multiple effects of AMSP induce excessive cellular S-nitrosylation, thereby guiding an untraditional apoptotic pathway, glyceraldehyde-3-phosphate dehydrogenase S-nitrosylation mediated nuclear translocation and protein degradation. Overall, our findings offer a new paradigm of homeostatic nanomedicine, potentially advancing oncotherapy modalities.

DNA Replication and Polymer Chain Duplication Reshape the Genome in Space and Time

In eukaryotes, DNA replication constitutes a complex process whereby multiple origins are stochastically fired, and from which the replication machinery proceeds along chromosomes to achieve the faithful synthesis of two identical copies of the genome during the S phase of the cell cycle. Experimental evidence shows a functional correlation between the dynamics of replication and the spatial organization of the genome inside cell nuclei, suggesting that the process of replicating DNA may impact chromosome folding. However, the theoretical and mechanistic bases of such a hypothesis remain elusive. To address that question, we propose a quantitative, minimal framework that integrates the dynamics of replication along a polymer chain by accounting explicitly for the progression of the replication machinery and the resulting formation of sister chromatids. By systematically characterizing the 3D structural consequences of replication, and of possible interactions between active replication machineries, we show that the formation of transient loops may potentially impact chromosome organization across multiple temporal and spatial scales, from the level of individual origins to that of the global polymer chain. Comparison with available microscopy and chromosome conformation capture data in yeast suggests that a replication-dependent loop extrusion process may be acting , and may shape chromosomes as loose polymer bottle brushes during the S phase. Lastly, we explore the postreplication relative organization of sister chromatids and demonstrate the emergence of catenations and intertwined structures, which are regulated by the density of fired origins. Published by the American Physical Society 2024

Mechanical Trapping of the Cell Nucleus Into Microgroove Concavity But Not On Convexity Induces Cell Tissue Growth and Vascular Smooth Muscle Differentiation

Mechanical Trapping of the Cell Nucleus Into Microgroove Concavity But Not On Convexity Induces Cell Tissue Growth and Vascular Smooth Muscle Differentiation

In Situ Oxygen Generation via a Platinum-Based Wireless Nanopore Electrode for Single-Cell Manipulation.

Oxygen production within human cells plays a critical role in cellular metabolism and is implicated in various diseases, including cancer. Investigating cellular heterogeneity under oxygen stimulation is crucial for elucidating disease mechanisms and advancing early therapeutic design. In this study, the platinum-based wireless nanopore electrode (WNE) with a diameter of ≈200nm is employed as a powerful tool to produce oxygen molecules near the cell nucleus. The oxygen production can be quantitatively controlled by adjusting the applied voltage. Through delivering oxygen near the cancer cell nucleus, this technique shows the capacity to alleviate the hypoxia microenvironment, a key factor in chemotherapy resistance. Furthermore, by modulating oxygen levels within individual living cells and delivering chemotherapeutic agents to the cancer cell nucleus, this approach offers significant potential for single-cell manipulation and the investigation of cellular heterogeneity under oxygen stimulation.

Modular Nanotransporters Deliver Anti-Keap1 Monobody into Mouse Hepatocytes, Thereby Inhibiting Production of Reactive Oxygen Species.

Background/Objectives: The study of oxidative stress in cells and ways to prevent it attract increasing attention. Antioxidant defense of cells can be activated by releasing the transcription factor Nrf2 from a complex with Keap1, its inhibitor protein. The aim of the work was to study the effect of the modular nanotransporter (MNT) carrying an R1 anti-Keap1 monobody (MNTR1) on cell homeostasis. Methods: The murine hepatocyte AML12 cells were used for the study. The interaction of fluorescently labeled MNTR1 with Keap1 fused to hrGFP was studied using the Fluorescence-Lifetime Imaging Microscopy-Förster Resonance Energy Transfer (FLIM-FRET) technique on living AML12 cells transfected with the Keap1-hrGFP gene. The release of Nrf2 from the complex with Keap1 and its levels in the cytoplasm and nuclei of the AML12 cells were examined using a cellular thermal shift assay (CETSA) and confocal laser scanning microscopy, respectively. The effect of MNT on the formation of reactive oxygen species was studied by flow cytometry using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. Results: MNTR1 is able to interact with Keap1 in the cytoplasm, leading to the release of Nrf2 from the complex with Keap1 and a rapid rise in Nrf2 levels both in the cytoplasm and nuclei, ultimately causing protection of cells from the action of hydrogen peroxide. The possibility of cleavage of the monobody in endosomes leads to an increase in the observed effects. Conclusions: These findings open up a new approach to specifically modulating the interaction of intracellular proteins, as demonstrated by the example of the Keap1-Nrf2 system.

Indigo alleviates psoriasis through the AhR/NF-κB signaling pathway: an in vitro and in vivo study.

Psoriasis is a chronic inflammatory skin disease. A strong association between the AhR/ NFκB axis and the inflammatory response in psoriasis. Indigo (IDG) has demonstrated significant anti-inflammatory properties. This study aimed to assess the anti-psoriatic efficacy of IDG while investigating the underlying mechanisms involved. In the in vitro experiments, cell viability was assessed using the CCK-8. qRT-PCR was employed to measure the mRNA levels of NF-κB, TNF-α, IL-1β, AhR, and CYP1A1. Western blotting was conducted to examine alterations in cytoplasmic and nuclear AhR protein levels. Additionally, an IDG nanoemulsion (NE) cream was prepared for the in vivo experiments. A psoriasis-like skin lesion mice model was induced using IMQ (62.5 mg/day for 7 days). The severity of psoriasis was evaluated using PASI, and skin lesions were scored while epidermal thickness was assessed via HE staining. The expression of inflammatory markers, including IL-6, IL-13, IL-17A, MCP-1, and TNF-α, was detected in skin lesions using Luminex. The levels of CYP1A1, p65, and p-p65 proteins were determined by Western blotting. LPS stimulation significantly elevated TNF-α, IL-6, and NF-κB mRNA levels, which were notably reduced by IDG treatment. Additionally, IDG significantly enhanced the expression of AhR and CYP1A1 mRNA. Further investigation revealed that IDG facilitated AhR translocation from the cytoplasm to the nucleus. In the IMQ-induced psoriasis-like mouse model, IDG NE substantially ameliorated the severity of skin lesions. Moreover, IDG NE treatment reduced the upregulation of inflammatory cytokines such as IL-6, IL-17A, MCP-1, and TNF-α in IMQ-induced skin lesions. It was also observed that IDG NE treatment increased CYP1A1 protein expression while inhibiting p65 and p-p65 protein expression. IDG emerges as a promising treatment for psoriasis, demonstrating effective therapeutic outcomes. Its mechanism of action is likely linked to the modulation of the AhR/NFκB signaling pathway.

Fgk3, a Glycogen Synthase Kinase, Regulates Chitin Synthesis through the Carbon Catabolite Repressor FgCreA in Fusarium graminearum.

The glycogen synthase kinase-3 (GSK3) orthologs are well-conserved in eukaryotic organisms. However, their functions remain poorly characterized in filamentous fungi. In our previous study, we unveiled the function of Fgk3, the GSK3 ortholog, in glycogen metabolism in Fusarium graminearum, the causal agent of Fusarium head blight. Interestingly, the fgk3 mutant was unstable and tended to produce fast-growing suppressors, including secondary suppressors. Using whole-genome sequencing, we identified suppressor mutations in FgCHS5, FgFKS1, FgCREA, FgSSN6, FgRGR1, and FgPP2A in nine primary and four secondary suppressors. Subsequently, we validated that deletion of FgCHS5 or FgCREAΔH253 mutation partially suppressed the defects of fgk3 in vegetative growth and cell wall integrity, suggesting that Fgk3 may regulate the chitin synthesis through FgCreA-mediated transcriptional regulation in F. graminearum. Accordingly, the FGK3 deletion led to hyphal swelling with abnormal chitin deposition, and deletion of FGK3 or FgCREA caused the upregulation of the expression of chitin synthases FgCHS5 and FgCHS6. The interaction between Fgk3 and FgCreA was verified by Yeast two-hybrid and Co-Immunoprecipitation assays. More importantly, we verified that the nuclear localization and protein stability of FgCreA relies on the Fgk3 kinase, while the H253 deletion facilitated the re-localization of FgCreA to the nucleus in the fgk3 mutant background, potentially contributing to the suppression of the fgk3 mutant's defects. Intriguingly, the ΔH253 mutation of FgCreA, identified in suppressor mutant S3, is adjacent to a conserved phosphorylation site, S254, suggesting that this mutation may inhibit the S254 phosphorylation and promote the nuclear localization of FgCreA. Collectively, our findings indicate that the glycogen synthase kinase Fgk3 regulates the chitin synthesis through the carbon catabolite repressor FgCreA in F. graminearum.

HIP is involved in NaCl and endoplasmic reticulum stress resistance in Arabidopsis

Heat shock protein 70 (HSP70)-interacting proteins (HIPs) have been studied in animals. HIPs perform diverse cellular functions, ranging from alleviating stress to suppressing the formation of insoluble protein, but how their orthologs function in plants is less known. This study shows that Arabidopsis HIP is a cytosolic and nuclear protein associated with HSP70. The hip mutants showed compromised tolerance to NaCl and endoplasmic reticulum (ER) stress, although they grew normally under standard growth conditions. Furthermore, hip mutants presented a decreased HSP70 level compared with the wild type under NaCl and ER stress conditions. These findings suggest the involvement of HIP in NaCl and ER stress tolerance.

CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB-specific cytoplasmic degradation. By adding a neuronal-specific NLS, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo postmitotic cell models.

How the Oocyte Nucleolus Is Turned into a Karyosphere: The Role of Heterochromatin and Structural Proteins.

Oocyte meiotic maturation includes large-scale chromatin remodeling as well as cytoskeleton and nuclear envelope rearrangements. This review addresses the dynamics of key cytoskeletal proteins (tubulin, actin, vimentin, and cytokeratins) and nuclear envelope proteins (lamin A/C, lamin B, and the nucleoporin Nup160) in parallel with chromatin reorganization in maturing mouse oocytes. A major feature of this reorganization is the concentration of heterochromatin into a spherical perinucleolar rim called surrounded nucleolus or karyosphere. In early germinal vesicle (GV) oocytes with non-surrounded nucleolus (without karyosphere), lamins and Nup160 are at the nuclear envelope while cytoplasmic cytoskeletal proteins are outside the nucleus. At the beginning of karyosphere formation, lamins and Nup160 follow the heterochromatin relocation assembling a new spherical structure in the GV. In late GV oocytes with surrounded nucleolus (fully formed karyosphere), the nuclear envelope gradually loses its integrity and cytoplasmic cytoskeletal proteins enter the nucleus. At germinal vesicle breakdown, lamin B occupies the karyosphere interior while all the other proteins stay at the karyosphere border or connect to chromatin. In metaphase oocytes, lamin A/C surrounds the spindle, Nup160 localizes to its poles, actin and lamin B are attached to the spindle fibers, and cytoplasmic intermediate filaments associate with both the spindle fibers and the metaphase chromosomes.

Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein involved in DNA repair, chromatin structure, and transcription. However, the regulation of its different functions remains poorly understood. Here, we report the role of PARP1 acetylation status in modulating its DNA repair and transactivation functions. We demonstrate that histone deacetylase 5 (HDAC5) determines PARP1 acetylation at Lys498 and Lys521 sites. HDAC5-mediated deacetylation at Lys498 site regulates PARP1 DNA damage response and facilitates efficient recruitment of DNA repair factors at damaged sites, thereby promoting cell survival. Additionally, HDAC5-mediated deacetylation at Lys521 site promotes PARP1 coactivator function, resulting in induction of proliferative and metabolic genes in an activating transcription factor 4-dependent manner. Thus, PARP1 induces metabolic adaptation to spur malignant phenotype. Our studies in mouse tumor models suggest that pharmacological inhibition of PARP1 enzymatic activity does not block tumor progression robustly as transactivation function remains unperturbed. These findings provide key mechanistic insights into PARP1 regulation and expand its role in tumor development.

CqProfilin enhances WSSV infection by promoting viral intracellular transport through binding to both viral nucleocapsid and actin cytoskeleton

White spot syndrome virus (WSSV) is a large nuclear-replicating DNA virus of crustaceans such as shrimp and crayfish; however, the molecular mechanisms facilitating its transport from the invasion site to the cell nucleus have not yet been well elucidated. In this study, a CqProfilin (CqPFN) with a conserved PROF domain was identified from the red claw crayfish Cherax quadricarinatus. CqPFN was ubiquitously expressed in all examined tissues and hemocyte, with the highest levels in the hemocyte, followed by hematopoietic tissue (Hpt) from which the hemocyte were derived in crayfish. The transcript of WSSV genes such as IE1 and VP28 was obviously decreased both in vivo in hemocyte and Hpt, as well as in vitro in cultured Hpt cells, after CqPFN gene silencing; in contrast, the expression of viral genes was significantly increased by the introduction of a recombinant CqPFN protein in Hpt cells in vitro. Moreover, CqPFN was clearly colocalized with the main viral nucleocapsid protein VP664 and F-actin cytoskeleton, respectively, during the early stage of WSSV infection in Hpt cells. In addition, CqPFN was confirmed to interact with a truncated VP6642,405-2,535 and another viral nucleocapsid protein VP15 of WSSV and Cqβ-Actin from Hpt by co-immunoprecipitation assays. Further studies found that VP664 also colocalized with F-actin in the Hpt cell cytoplasm after WSSV infection, suggesting that the actin cytoskeleton was involved in the intracellular transport of incoming viral nucleocapsid. Taken together, CqPFN might combine with the actin cytoskeleton to promote WSSV infection through binding with viral nucleocapsid proteins VP664 and VP15, promoting intracellular transport of viral incoming nucleocapsid for further releasing genome into the nucleus for transcription. Collectively, these results provided an understanding of the WSSV pathogenesis, which will contribute to the development of an antiviral strategy against WSSV disease.

RHBDF1 promotes PERK expression through the JNK/FoxO3 pathway in breast cancer cells.

Human rhomboid family-1 ( RHBDF1) gene is recognized as an oncogene involved in breast cancer development. Previous studies have indicated that RHBDF1 contributes significantly to endoplasmic reticulum (ER) protein homeostasis by stabilizing the binding immunoglobulin protein (BiP) and promoting the unfolded protein response (UPR). Here, we report a relationship between RHBDF1 and the ER stress sensors PERK, IRE1, and ATF6. We show that RHBDF1 deficiency in breast cancer cells results in decreased levels of PERK, pPERK, and peIF2α. These protein levels can be restored in RHBDF1-deficient breast cancer cells by artificial overexpression of RHBDF1 but not IRE1 or ATF6. Additionally, we show that the transcription factor FoxO3 is essential for the RHBDF1-mediated production of PERK. Subsequent analysis reveals that RHBDF1 activates JNK, which causes FoxO3 to translocate into the cell nucleus. These findings demonstrate that RHBDF1 supports the UPR by upregulating the PERK/peIF2α pathway via the JNK/FoxO3 axis and that the functions of RHBDF1 are essential for preserving the homeostasis of ER proteins.

Dissociation of the nuclear WWOX/TRAF2 switch renders UV/cold shock-mediated nuclear bubbling cell death at low temperatures

BackgroundNormal cells express functional tumor suppressor WW domain-containing oxidoreductase (WWOX), designated WWOXf. UV irradiation induces WWOXf cells to undergo bubbling cell death (BCD) — an event due to the accumulation of nuclear nitric oxide (NO) gas that forcefully pushes the nuclear and cell membranes to form one or two bubbles at room temperature (22 °C) and below. In contrast, when WWOX-deficient or -dysfunctional (WWOXd) cells are exposed to UV and/or cold shock, the cells undergo nuclear pop-out explosion death (POD). We aimed to determine the morphological and biochemical changes in WWOXf cells during BCD versus apoptosis.MethodsWWOXf and WWOXd cells were exposed to UV followed by measuring BCD or POD by time-lapse microscopy and/or time-lapse holographic microscopy at 4, 22, or 37 °C to visualize morphological changes. Live cell stains were used to measure the kinetics of nitric oxide (NO) production and Ca2+ influx. Extent of cell death was measured by uptake of propidium iodide and by internucleosomal DNA fragmentation using agarose gel electrophoresis.ResultsWWOXf cells were exposed to UV and then cold shock, or cold shock and then UV, and cultured at 4, 10, and 22 °C, respectively. Initially, UV induced calcium influx and NO production, which led to nuclear bubbling and final death. Cold shock pretreatment completely suppressed UV-mediated bubbling at 37 °C, so the UV/cold shock-treated cells underwent apoptosis. Without cold shock, UV only induced bubbling at all temperatures, whereas the efficiency of bubbling at 37 °C was reduced by greater than 50%. Morphologically, the WWOXf cell height or thickness was significantly increased during cell division or apoptosis, but the event did not occur in BCD. In comparison, when WWOXd cancer cells received UV or UV/cold shock, these cells underwent NO-independent POD. UV/cold shock effectively downregulated the expression of many proteins such as the housekeeping α-tubulin (> 70%) and β-actin (< 50%), and cortactin (> 70%) in WWOXf COS7 cells. UV/cold shock induced relocation of α-tubulin to the nucleus and nuclear bubbles in damaged cells. UV induced co-translocation of the WWOX/TRAF2 complex to the nuclei, in which the prosurvival TRAF2 blocked the proapoptotic WWOX via its zinc finger domain. Without WWOX, TRAF2 did not relocate to the nuclei. Cold shock caused the dissociation of the WWOX/TRAF2 complex in the nucleus needed for BCD. In contrast, the formation of the WWOX/TRAF2 complex, plus p53, was strengthened at 37 °C required for apoptosis.ConclusionsThe temperature-sensitive nuclear WWOX/TRAF2 complex acts as a molecular switch, whose dissociation favors BCD at low temperatures, and the association supports apoptosis at 37 °C in UV-treated WWOXf cells.

SON-dependent nuclear speckle rejuvenation alleviates proteinopathies.

Current treatments targeting individual protein quality control have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common upstream druggable target capable of global proteostasis modulation. Building on our prior research establishing nuclear speckles as a pivotal membrane-less organelle responsible for global proteostasis transcriptional control, we aim to alleviate proteinopathies through nuclear speckle rejuvenation. We identified pyrvinium pamoate as a small-molecule nuclear speckle rejuvenator that enhances protein quality control while suppressing YAP1 signaling via decreasing the surface/interfacial tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, nanomolar pyrvinium pamoate alleviated retina degeneration and reduced tauopathy by promoting autophagy and ubiquitin-proteasome system in a SON-dependent manner without causing cellular stress. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity were also observed in human tauopathies. Our study uncovers novel therapeutic targets for tackling protein misfolding disorders within an expanded proteostasis framework encompassing nuclear speckles and YAP1.

SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing.

O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a 'decoy exon' present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.

P02.15.A INVESTIGATING THE ROLE OF CELLULAR SENESCENCE IN BRIDGING GLIOBLASTOMA AND ALZHEIMER’S DISEASE

Abstract BACKGROUND With changing demographics, age-related diseases like neurodegenerative disorders and cancer are becoming increasingly prevalent. We previously demonstrated that over 50% of glioblastoma (GB) patients show Alzheimer’s Disease (AD)-like changes in the tumor-adjacent cortex. However, whether these diseases are biologically linked remains unclear. Here, we investigate cellular senescence, a hallmark of aging, as a shared mechanism. MATERIAL AND METHODS We utilized single cell and single nucleus RNA sequencing datasets from 110 GB patients (GBmap core atlas) and 89 individuals with varying stages of AD (SEA-AD MTG atlas). Following data integration, we assessed senescent cells using three established gene lists, performed differential gene expression and conducted gene set enrichment analysis. Senescence scores were compared across different cell types and patients. Co-expression modules with senescence-associated genes were identified using weighted gene co-expression network analysis (WGCNA). Quantitative neuropathological analysis of published measurements (Iba1, AT8, 6E10) complemented the molecular findings. RESULTS Senescence was found in both GB and AD patient cells including cell types of the microenvironment. Notably, the variability in scores per patient was significantly higher in GB as compared to AD cell types, indicating greater inter-patient heterogeneity. Microglia were specifically enriched for senescence gene sets among glial cells. Subset analysis of microglia through data harmonization identified a proinflammatory GB microglia subtype that shared high similarity with AD microglia and had highest senescence scores. Notably, the increase in senescent microglia was associated with progression to advanced disease stages in both diseases. WGCNA identified 3 consensus modules of senescence-associated genes that were coexpressed with disease-specific genes, like PTEN implicated phosphorylation in the AD module and SPP1 for extracellular matrix remodeling in the GB module. All senescence associated modules showed increased expression in more advanced disease stages. Quantitative analysis of immunohistochemistry measurements in the AD dataset confirmed that samples with a high senescence score showed a significantly higher density of tau pathology and an altered microglia morphology. CONCLUSION Our study identifies cellular senescence as one connection between GB and AD pathology by utilizing atlas-level transcriptomic datasets. Particularly microglia upregulate senescence signatures with disease progression. The co-expression of senescence-associated genes within disease-specific modules underscores the complexity of senescence in this context.