Nuclear Localization Research Articles

Phosphate-dependent nuclear export via a novel NES class recognized by exportin Msn5.

Gene expression in response to environmental stimuli is dependent on nuclear localization of key signaling components, which can be tightly regulated by phosphorylation. This is exemplified by the phosphate-sensing transcription factor Pho4, which requires phosphorylation for nuclear export by the yeast exportin Msn5. Unlike the traditional hydrophobic nuclear export signal (NES) utilized by the Exportin-1/XPO1 system, cryogenic-electron microscopy structures reveal that Pho4 presents a novel, phosphorylated 35-residue NES that interacts with the concave surface of Msn5 through two Pho4 phospho-serines that align with two Msn5 basic patches, unveiling a previously unknown mechanism of phosphate-specific recognition. Furthermore, the discovery that unliganded Msn5 is autoinhibited explains the positive cooperativity of Pho4/Ran-binding and proposes a mechanism for Pho4's release in the cytoplasm. These findings advance our understanding of the diversity of signals that drive nuclear export and how cargo phosphorylation is crucial in regulating nuclear transport and controlling cellular signaling pathways.

SIRT2 regulates high mobility group protein B1 nucleoplasmic shuttle and degradation via deacetylation in microglia.

High mobility group protein B1 (HMGB1) acts as a pathogenic inflammatory response to mediate ranges of conditions such as epilepsy, septic shock, ischemia, traumatic brain injury, Parkinson's disease, Alzheimer's disease and mass spectrometry. HMGB1 promotes inflammation during sterile and infectious damage and plays a crucial role in disease development. Mobilization from the nucleus to the cytoplasm is the first important step in the release of HMGB1 from activated immune cells. Here, we demonstrated that Sirtuin 2 (SIRT2) physically interacts with and deacetylates HMGB1 at 43 lysine residue at nuclear localization signal locations, strengthening its interaction with HMGB1 and causing HMGB1 to be localized in the cytoplasm. These discoveries are the first to shed light on the SIRT2 nucleoplasmic shuttle, which influences HMGB1 and its degradation, hence revealing novel therapeutic targets and avenues for neuroinflammation treatment.

Proximity labelling of pro-interleukin-1α reveals evolutionary conserved nuclear interactions.

Interleukin-1α is a suggested dual-function cytokine that diverged from interleukin-1β in mammals potentially by acquiring additional biological roles that relate to highly conserved regions in the pro-domain of interleukin-1α, including a nuclear localisation sequence and histone acetyltransferase-binding domains. Why evolution modified pro-interleukin-1α's subcellular location and protein interactome, and how this shaped interleukin-1α's intracellular role, is unknown. Here we show that TurboID proximity labelling with pro-interleukin-1α suggests a nuclear role for pro-interleukin-1α that involves interaction with histone acetyltransferases, including EP300. We also identify and validate inactivating mutations in the pro-interleukin-1α nuclear localisation sequence of multiple mammalian species, including toothed whales, castorimorpha and marsupials. However, histone acetyltransferase-binding domains are conserved in those species that have lost pro-interleukin-1α nuclear localisation. Together, these data suggest that histone acetyltransferase binding and nuclear localisation occurred together, and that while some species lost the nuclear localisation sequence in their pro-interleukin-1α, histone acetyltransferase binding ability was maintained. The nuclear localisation sequence was lost from several distinct species at different evolutionary times, suggesting convergent evolution, and that the loss of the nuclear localisation sequence confers some important biological outcome.

A Combined Transcriptomic and Proteomic Analysis of Monkeypox Virus A23 Protein on HEK293T Cells.

Monkeypox virus (MPXV) is a cross-kingdom pathogen infecting both humans and wildlife, which poses a significant health risk to the public. Although MPXV attracts broad attention, there is a lack of adequate studies to elucidate pathogenic mechanisms associated with viral infections. In this study, a high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to explore the transcriptional and metabolic responses of MPXV A23 protein to HEK293T cells. The protein-protein interactions and signaling pathways were conducted by GO and KEGG analyses. The localization of A23 protein in HEK293T cells was detected by immunofluorescence. A total of 648 differentially expressed genes (DEGs) were identified in cells by RNA-Seq, including 314 upregulated genes and 334 downregulated genes. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected 115 cellular proteins that interact with the A23 proteins. Transcriptomic sequencing analysis revealed that transfection of MPXV A23 protein modulated genes primarily associated with cellular apoptosis and DNA damage repair. Proteomic analysis indicated that this protein primarily interacted with host ribosomal proteins and histones. Following the identification of the nuclear localization sequence RKKR within the A23 protein, a truncated mutant A23ΔRKKR was constructed to investigate the subcellular localization of A23 protein. The wild-type A23 protein exhibits a significantly higher nuclear-to-cytoplasmic ratio, exceeding 1.5, in contrast to the mutant A23ΔRKKR, which has a ratio of approximately 1. Immunofluorescence assays showed that the A23 protein was mainly localized in the nucleus. The integration of transcriptomics and proteomics analysis provides a comprehensive understanding of the interaction between MPXV A23 protein and the host. Our findings highlight the potential role of this enzyme in suppressing host antiviral immune responses and modulating host gene expression.

Targeting adenocarcinoma and enzalutamide‑resistant prostate cancer using the novel anti‑androgen inhibitor ADA‑308.

Prostate cancer (PCa) is the leading cause of cancer‑related death among men worldwide. PCa often develops resistance to standard androgen deprivation therapy and androgen receptor (AR) pathway inhibitors, such as enzalutamide (ENZ). Therefore, there is an urgent need to develop novel therapeutic strategies for this disease. The efficacy of ADA‑308 was evaluated through in vitro assessments of AR activity and cell proliferation, alongside in vivo studies. ADA‑308 has emerged as a promising candidate, demonstrating potent inhibition of AR‑sensitive adenocarcinoma as well as ENZ‑resistant PCa cell lines. The results of the study revealed that ADA‑308 effectively blocked AR activity, including its nuclear localization, and inhibited cell proliferation in vitro. Furthermore, ADA‑308 demonstrated notable efficacy in vivo, with a robust antitumor response in ENZ‑resistant models. These findings establish the role of ADA‑308 as a potent AR inhibitor that overcomes resistance to AR‑targeted therapies and highlights its potential as a novel therapeutic approach in advanced PCa management.

Peroxiredoxin 2 regulates DAF-16/FOXO mediated mitochondrial remodelling in response to exercise that is disrupted in ageing

ObjectivesA decline in mitochondrial function and increased susceptibility to oxidative stress is a hallmark of ageing. Exercise endogenously generates reactive oxygen species (ROS) in skeletal muscle and promotes mitochondrial remodelling resulting in improved mitochondrial function. It is unclear how exercise induced redox signalling results in alterations in mitochondrial dynamics and morphology. MethodsIn this study, a Caenorhabditis elegans model of exercise and ageing was used to determine the mechanistic role of Peroxiredoxin 2 (PRDX-2) in regulating mitochondrial morphology. Mitochondrial morphology was analysed using transgenic reporter strains and transmission electron microscopy, complimented with the analysis of the effects of ageing and exercise on physiological activity. ResultsThe redox state of PRDX-2 was altered with exercise and ageing, hyperoxidised peroxiredoxins were detected in old worms along with basally elevated intracellular ROS. Exercise generated intracellular ROS and rapid mitochondrial remodelling, which was disrupted with age. The exercise intervention promoted mitochondrial ER contact sites (MERCS) assembly and increased DAF-16/FOXO nuclear localisation. The prdx-2 mutant strain had a disrupted mitochondrial network as evidenced by increased mitochondrial fragmentation. In the prdx-2 mutant strain, exercise did not activate DAF-16/FOXO, mitophagy or increase MERCS assembly. The results demonstrate that exercise generated ROS increased DAF-16/FOXO transcription factor nuclear localisation required for activation of mitochondrial fusion events that were blunted with age. ConclusionsThe data demonstrate the critical role of PRDX-2 in orchestrating mitochondrial remodelling in response to a physiological stress by regulating redox dependent DAF-16/FOXO nuclear localisation.

PTEN regulated by gga-miR-20a-5p is involved in chicken macrophages inflammatory response to APEC infection via autophagy

Colibacillosis, a bacterial disease caused by avian pathogenic E. coli (APEC), is a prevalent condition in the poultry industry, resulting in substantial economic losses annually. Previously, we identified PTEN as a crucial candidate gene that may play a significant role in chicken's immune response to APEC infection. Bioinformatics analysis indicated that the PTEN protein was unstable, hydrophilic and nuclear localization, with multiple putative phosphorylation sites and a high degree of similarity to duck and goose PTEN. Moreover, PTEN exhibited high expression levels in various tissues such as the stomach, cecum, small intestine, spleen, thymus, harderian gland, muscle, cerebrum, cerebellum, lung, and liver in comparison to heart tissue. Overexpression of PTEN resulted in a significant promotion of the expression level of pro-apoptosis genes and inflammatory mediators, as well as the production of NO, with or without APEC infection, which led to cellular injury. Furthermore, overexpression of PTEN was found to regulate the expression levels of autophagy related genes, regardless of APEC infection. Additionally, PTEN was a target gene of gga-miR-20a-5p and regulated by gga-miR-20a-5p upon APEC infection. Taken together, these findings establish a foundation for investigating the biological function of chicken PTEN, providing a potential target for future treatments against APEC infection as well as the breeding of genetically resistant poultry.

Mechanisms for controlling Dorsal nuclear levels.

Formation of the Dorsal nuclear-cytoplasmic gradient is important for the proper establishment of gene expression patterns along the dorsal-ventral (DV) axis during embryogenesis in Drosophila melanogaster. Correct patterning of the DV axis leads to formation of the presumptive mesoderm, neurogenic ectoderm, dorsal ectoderm, and amnioserosa, which are tissues necessary for embryo viability. While Toll signaling is necessary for Dorsal gradient formation, a gradient still forms in the absence of Toll, suggesting there are additional mechanisms required to achieve correct nuclear Dorsal levels. Potential mechanisms include post-translational modification, shuttling, and nuclear spacing. Post-translational modification could affect import and export rates either directly through modification of a nuclear localization sequence or nuclear export sequence, or indirectly by affecting interactions with binding partners that alter import and export rates. Shuttling, which refers to the facilitated diffusion of Dorsal through its interaction with its cytoplasmic inhibitor Cactus, could regulate nuclear levels by delivering more Dorsal ventrally. Finally, nuclear spacing could result in higher nuclear levels by leaving fewer nuclei in the ventral domain to uptake Dorsal. This review details how each of these mechanisms may help establish Dorsal nuclear levels in the early fly embryo, which serves as a paradigm for understanding how the dynamics of graded inputs can influence patterning and target gene expression. Furthermore, careful analysis of nuclear Dorsal levels is likely to provide general insights as recent studies have suggested that the regulation of nuclear import affects the timing of gene expression at the maternal-to-zygotic transition.

Hypoxia-inducible factors in postnatal mouse molar dental pulp development: insights into expression patterns, localisation and metabolic pathways.

Hypoxia is relevant to several physiological and pathological processesand this also applies for the tooth. The adaptive response to lowering oxygen concentration is mediated by hypoxia-inducible factors (HIFs). Since HIFs were shown to participate in the promotion of angiogenesis, stem cell survival, odontoblast differentiation and dentin formation, they may play a beneficial role in the tooth reparative processes. Although some data were generated in vitro, little is known about the in vivo context of HIFs in tooth development. In order to contribute to this field, the mouse mandibular first molar was used as a model.The expression and in situ localisation of HIFs were examined at postnatal (P) days P0, P7, P14, using RT-PCR and immunostaining. The expression pattern of a broad spectrum of hypoxia-related genes was monitored by customised PCR Arrays. Metabolic aspects were evaluated by determination of the lactate level and mRNA expression of the mitochondrial marker Nd1.The results show constant high mRNA expression of Hif1a, increasing expression of Hif2a, and very low expression of Hif3a during early postnatal molar development. In the examined period the localisation of HIFs in the nuclei of odontoblasts and the subodontoblastic layer identified their presence during odontoblastic differentiation. Additionally, the lower lactate level and higher expression of mitochondrial Nd1 in advanced development points to decreasing glycolysis during differentiation. Postnatal nuclear localisation of HIFs indicates a hypoxic state in specific areas of dental pulp as oxygen demands depend on physiological events such as crown and root dentin mineralization.

Hydrostatic pressure drives sprouting angiogenesis via adherens junction remodelling and YAP signalling

Endothelial cell physiology is governed by its unique microenvironment at the interface between blood and tissue. A major contributor to the endothelial biophysical environment is blood hydrostatic pressure, which in mechanical terms applies isotropic compressive stress on the cells. While other mechanical factors, such as shear stress and circumferential stretch, have been extensively studied, little is known about the role of hydrostatic pressure in the regulation of endothelial cell behavior. Here we show that hydrostatic pressure triggers partial and transient endothelial-to-mesenchymal transition in endothelial monolayers of different vascular beds. Values mimicking microvascular pressure environments promote proliferative and migratory behavior and impair barrier properties that are characteristic of a mesenchymal transition, resulting in increased sprouting angiogenesis in 3D organotypic model systems ex vivo and in vitro. Mechanistically, this response is linked to differential cadherin expression at the adherens junctions, and to an increased YAP expression, nuclear localization, and transcriptional activity. Inhibition of YAP transcriptional activity prevents pressure-induced sprouting angiogenesis. Together, this work establishes hydrostatic pressure as a key modulator of endothelial homeostasis and as a crucial component of the endothelial mechanical niche.

Abstract Tu058: Reverse Remodeling with LVAD Therapy is Associated with Alternative Splicing of CAMK2D

Therapies for end-stage heart failure are lacking and the number of patients awaiting heart transplants far outweighs availability. Left ventricular assist devices (LVADs) take over cardiac pump function and are used as a bridge-to-transplant. Notably, some patients on LVADs experience recovery of cardiac function and reversal of pathological cardiac remodeling; however, the molecular mechanisms underlying these favorable outcomes are unknown. In this study, we studied 19 paired pre- and post-LVAD samples with 10 clinically categorized as responders and 9 as non-responders. Bulk mRNA sequencing identified 486 differentially expressed genes post-LVAD, including reduced pro-inflammatory gene expression and increased expression of genes involved in metal ion binding; however, just 23 total genes were differentially expressed between responders and non-responders. To determine whether LVAD differentially impacted the proteome in responders and non-responders, we next performed quantitative proteomics on post-LVAD samples. This analysis identified 154 differentially expressed proteins and revealed that factors involved in mRNA processing were higher expressed in LVAD responders. Re-analysis of our mRNA sequencing data for gene isoforms identified that alternative splicing of calcium and calmodulin-dependent protein kinase 2-delta ( CAMK2D ) was the most significantly impacted event with non-responders having increased inclusion of exon 14 (denoting the B isoform), which encodes a nuclear localization sequence. Notably, there was a strong negative correlation between exon 14 inclusion and ΔLVEF post-LVAD. We validated these findings using targeted exon-specific quantitative PCR, which confirmed that expression of CAMK2D B was higher both pre- and post-LVAD in non-responders, while responders had post-LVAD levels matching non-failing controls. We next investigated whether responders and non-responders differed at the phospho-proteome level via mass spectrometry and found three serines in CaMK2δ exon 14 were the only differentially abundant phosphosites and all increased in non-responders. Localization studies in vitro with adenoviruses expressing phospho-null or phospho-mimetic CaMK2D B constructs revealed that these phosphorylation events precluded nuclear localization. In conclusion, we identified differential alternative splicing and phosphorylation of CaMK2δ in LVAD responders, which may represent novel therapeutic targets for heart failure recovery.

Abstract We001: Doxorubicin-Induced hiPSC Hormetic Model

Doxorubicin (Dox), an anticancer anthracycline drug, is an important tool to understand how cells develop resistance to anthracyclines. Hormesis is a biological phenomenon where low doses of drugs can have beneficial effects on cell proliferation and differentiation. hiPSCs are becoming the model of choice for Dox studies yet the low dose hormetic effect of Dox on hiPSC proliferation and differentiation is not well understood. We assess the hypothesis that low-dose Dox preconditioning of hiPSCs enhances pluripotency gene expression while maintaining pluripotency state. To determine the low-dose effect (50 nM), we treated the JHU001 hiPSC line with Dox (hiPSC-D50) for two hours and a vehicle control without Dox (hiPSC-D0). Both hiPSC-D50 and hiPSC-D0 were maintained in E8 flex medium culture until reaching 70% confluency and were passaged five times and frozen for experimental studies. hiPSC-D50 and -D0 were thawed at passage 5 and cultured on coverslips for pluripotent gene expression (Oct-4, Nanog, and SSEA-4) analysis by immunocytochemistry. The cells were imaged using epifluorescence Nikon Eclipse Ti2 microscopy and the images were analyzed using NIS-Elements BR software. Phase-contrast microscopy shows significant cell death in hiPSC-D50 24 hours post-treatment compared to hiPSC-D0. However, 72 hours post-Dox-treatment, several colonies with pluripotent morphology were observed in hiPSC-D50 that were similar to hiPSC-D0. While some differentiated cell morphologies were observed in hiPSC-D50, these were significantly reduced after five passages. Immunofluorescent staining of Oct-4 and Nanog showed nuclear localization whereas SSEA-4 was localized on the cell surface, in both hiPSC-D0 and hiPSC-D50 cells at passage five. However, immunocytochemistry analysis of pluripotent markers revealed significantly higher expression of Oct-4, Nanog and SSEA-4 in hiPSC-D50 when compared to hiPSC-D0 (1.3-fold,1.9-fold and 2.9-fold increase with p-values: 0.00057, 0.018, and 0.0000026, respectively; n= 5) suggesting a more robust pluripotent state and self-renewal capacity. Interestingly, higher SSEA-4 expression might also be associated with resistance to Dox. Our data suggest that low-dose Dox preconditioning enhances pluripotency state. This Dox-induced hormesis model of hiPSCs is a unique platform to help understand how Dox upregulate pluripotency markers and the potential link between SSEA-4 and drug resistance.

Notch1 Phase Separation Coupled Percolation facilitates target gene expression and enhancer looping.

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

Research on the anti-oxidant and anti-aging effects of Polygonatum kingianum saponins in Caenorhabditis elegans

Oxidative stress and its impact on aging are critical areas of research. Natural anti-oxidants, such as saponins found in Polygonatum sibiricum, hold promise as potential clinical interventions against aging. In this study, we utilized the nematode model organism, Caenorhabditis elegans, to investigate the pharmacological effects of Polygonatum sibiricum saponins (PKS) on antioxidation and anti-aging. The results demonstrated a significant anti-aging biological activity associated with PKS. Through experiments involving lifespan and stress, lipofuscin, q-PCR, and ROS measurement, we found that PKS effectively mitigated aging-related processes. Furthermore, the mechanism underlying these anti-aging effects was linked to the SKN-1 signaling pathway. PKS increased the nuclear localization of the SKN-1 transcription factor, leading to the up-regulation of downstream anti-oxidant genes, such as gst-4 and sod-3, and a substantial reduction in intracellular ROS levels within the nematode. In conclusion, our study sheds light on the anti-oxidant and anti-aging properties of PKS in C. elegans. This research not only contributes to understanding the biological mechanisms involved but also highlights the potential therapeutic applications of these natural compounds in combating aging-related processes.

A novel peptide encoded by circ-SLC9A6 promotes lipid dyshomeostasis through the regulation of H4K16ac-mediated CD36 transcription in NAFLD.

As the leading cause of end-stage liver disease, nonalcoholic fatty liver disease (NAFLD) is mainly induced by lipid dyshomeostasis. The translation of endogenous circular RNAs (circRNAs) is closely related to the progression of various diseases, but the involvement of circRNAs in NAFLD has not been determined. Combined high-throughput circRNA profiles were used to identify circRNAs with translational potential. The underlying molecular mechanisms were investigated by RNA sequencing, pull-down/MS and site-specific mutagenesis. In this study, we focused on circ-SLC9A6, an abnormally highly expressed circRNA in human and mouse liver tissue during NAFLD development that exacerbates metabolic dyshomeostasis in hepatocytes by encoding a novel peptide called SLC9A6-126aa in vivo and in vitro. YTHDF2-mediated degradation of m6A-modified circ-SLC9A6 was found to be essential for the regulation of SLC9A6-126aa expression. We further found that the phosphorylation of SLC9A6-126aa by AKT was crucial for its cytoplasmic localization and the maintenance of physiological homeostasis, whereas high-fat stress induced substantial translocation of unphosphorylated SLC9A6-126aa to the nucleus, resulting in a vicious cycle of lipid metabolic dysfunction. Nuclear SLC9A6-126aa promotes transcriptional activation of the target gene CD36 and enhances its occupancy of the CD36 promoter locus by regulating MOF-mediated histone H4K16 acetylation. Hepatic CD36 depletion significantly ameliorated hyperactivated MAPK signalling and lipid disturbance in SLC9A6-126aa transgenic mice. Clinically, increasing levels of SLC9A6-126aa were observed during NAFLD progression and were found to be positively correlated with the CD36 and MAPK cascades. This study revealed the role of circ-SLC9A6-derived SLC9A6-126aa in the epigenetic modification-mediated regulation of lipid metabolism. Our findings may provide promising therapeutic targets for NAFLD and new insights into the pathological mechanisms of metabolic diseases. Under normal circumstances, driven by m6A modification, YTHDF2 directly recognizes and degrades circ-SLC9A6, thereby inhibiting the translation of SLC9A6-126aa. Additionally, AKT1 phosphorylates and inhibits the nuclear translocation of SLC9A6-126aa. In NAFLD, lipid overload leads to YTHDF2 and AKT1 deficiency, ultimately increasing the expression and nuclear import of SLC9A6-126aa. Nuclear SLC9A6-126aa binds directly to the CD36 promoter and initiates CD36 transcription, which induces lipid dyshomeostasis.

Characterization of capsid protein of porcine circovirus 4 by in-silico approach

Porcine Circovirus 4 (PCV4) has been detected in pigs from China only in 2019. PCV4 has been found to be associated with several forms of clinical syndromes in pigs. Like other Porcine Circoviruses, the only structural protein of PCV4 is the capsid protein. Therefore, it could be a key vaccine candidate or diagnostic marker. The present study aims to characterize the capsid protein of PCV4 by using several bioinformatics tools. The in-silico analysis revealed that the molecular weight of PCV4 capsid protein is about 27.3 kDa which is made up of 228 amino acids. The capsid protein of PCV4 contains a nuclear localization signal at N-terminal and several post-translational modifications could be predicted at different residues of the protein. The secondary structure of capsid protein contains coil, beta strand, and alpha helix. The 3D structure of the protein was predicted with a high level of confidence. The capsid protein of PCV4 also contains several potential B-cell epitopes. Thus, the in-silico characterization of the capsid protein will be beneficial in designing diagnostics and vaccine candidates in future to combat PCV4 infection in pigs.

Effect of rhamnogalacturonan-I-rich polysaccharides isolated from crabapple hydrolysates on IL-1β-induced inflammation in intestinal epithelial cells

This study aimed to investigate the structural characteristics and intracellular mechanisms of polysaccharides (MP-PE-I) purified from a crabapple (Malus prunifolia) enzymatic hydrolysate (MP-PE). Activity-guided fractionation revealed that MP-PE-I was the active moiety and significantly reduced the production and gene expression of pro-inflammatory factors in interleukin (IL)-1β-treated intestinal epithelial cells (Caco-2). Moreover, MP-PE-I downregulated the phosphorylation and nuclear localization of proteins involved in the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways, as evidenced by immunoblotting and immunofluorescence analysis. In antagonistic studies with specific inhibitors of the MAPK and NF-κB pathways, IL-6 inhibition was significantly regulated by p38; IL-8 by IκBα, JNK, and p38; and monocyte chemoattractant protein-1 (MCP-1) by JNK, p38, and ERK. Additionally, MP-PE-I significantly decreased the mRNA and protein expression of IL-1 receptor type 1. Chemical and structural characteristic analyses showed that MP-PE-I is a polysaccharide rich in rhamnogalacturonan (RG)-I and plays a crucial role in intestinal immunomodulation. To our knowledge, this is the first study to demonstrate the intestinal immunomodulatory activity, intracellular mechanisms, and structural characteristics of RG-I-rich polysaccharides isolated from crabapples.

Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses.

Biotic and abiotic stresses have already seriously restricted the growth and development of Pinus massoniana, thereby influencing the quality and yield of its wood and turpentine. Recent studies have shown that C2H2 zinc finger protein transcription factors play an important role in biotic and abiotic stress response. However, the members and expression patterns of C2H2 TFs in response to stresses in P. massoniana have not been performed. In this paper, 57 C2H2 zinc finger proteins of P. massoniana were identified and divided into five subgroups according to a phylogenetic analysis. In addition, six Q-type PmC2H2-ZFPs containing the plant-specific motif 'QALGGH' were selected for further study under different stresses. The findings demonstrated that PmC2H2-ZFPs exhibit responsiveness towards various abiotic stresses, including drought, NaCl, ABA, PEG, H2O2, etc., as well as biotic stress caused by the pine wood nematode. In addition, PmC2H2-4 and PmC2H2-20 were nuclear localization proteins, and PmC2H2-20 was a transcriptional activator. PmC2H2-20 was selected as a potential transcriptional regulator in response to various stresses in P. massoniana. These findings laid a foundation for further study on the role of PmC2H2-ZFPs in stress tolerance.

Chemotherapy-induced acetylation of ACLY by NAT10 promotes its nuclear accumulation and acetyl-CoA production to drive chemoresistance in hepatocellular carcinoma

Chemotherapeutic efficacy is seriously impeded by chemoresistance in more than half of hepatocellular carcinoma (HCC) patients. However, the mechanisms involved in chemotherapy-induced upregulation of chemoresistant genes are not fully understood. Here, this study unravels a novel mechanism controlling nuclear acetyl-CoA production to activate the transcription of chemoresistant genes in HCC. NAT10 is upregulated in HCC tissues and its upregulation is correlated with poor prognosis of HCC patients. NAT10 is also upregulated in chemoresistant HCC cells. Targeting NAT10 increases the cytotoxicity of chemotherapy in HCC cells and mouse xenografts. Upon chemotherapy, NAT10 translocates from the nucleolus to the nucleus to activate the transcription of CYP2C9 and PIK3R1. Additionally, nuclear acetyl-CoA is specifically upregulated by NAT10. Mechanistically, NAT10 binds with ACLY in the nucleus and acetylates ACLY at K468 to counteract the SQSTM1-mediated degradation upon chemotherapy. ACLY K468-Ac specifically accumulates in the nucleus and increases nuclear acetyl-CoA production to activate the transcription of CYP2C9 and PIK3R1 through enhancing H3K27ac. Importantly, K468 is required for nuclear localization of ACLY. Significantly, ACLY K468-Ac is upregulated in HCC tissues, and ablation of ACLY K468-Ac sensitizes HCC cells and mouse xenografts to chemotherapy. Collectively, these findings identify NAT10 as a novel chemoresistant driver and the blockage of NAT10-mediated ACLY K468-Ac possesses the potential to attenuate HCC chemoresistance.

Autoregulatory mechanism of enzyme activity by the nuclear localization signal of lysine-specific demethylase 1

The N-terminal region of the human Lysine Specific Demethylase 1 (LSD1) has no predicted structural elements, contains a nuclear localization signal (NLS), undergoes multiple post-translational modifications (PTMs), and acts as a protein-protein interaction hub. This intrinsically disordered region (IDR) extends from core LSD1 structure, resides atop the catalytic active site, and is known to be dispensable for catalysis. Here, we show differential nucleosome binding between the full-length and an N-terminus deleted LSD1 and identify that a conserved NLS and PTM containing element of the N-terminus contains an alpha helical structure, and that this conserved element impacts demethylation. Enzyme assays reveal that LSD1’s own electropositive NLS amino acids 107-120 inhibit demethylation activity on a model Histone 3 lysine 4 di-methyl (H3K4me2) peptide (Kiapp ∼ 3.3 μM) and H3K4me2 nucleosome substrates (IC50 ∼ 30.4 μM), likely mimicking the histone H3 tail. Further, when the identical, inhibitory NLS region contains phosphomimetic modifications, inhibition is partially relieved. Based upon these results and biophysical data, a regulatory mechanism for the LSD1-catalyzed demethylation reaction is proposed whereby NLS-mediated autoinhibition can occur through electrostatic interactions, and be partially relieved through phosphorylation that occurs proximal to the NLS. Taken together, the results highlight a dynamic and synergistic role for PTMs, IDRs, and structured regions near LSD1 active site and introduces the notion that phosphorylated mediated NLS regions can function to fine-tune chromatin modifying enzyme activity.