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Protein phosphorylation, a widely occurring and significant post-translational modification, is integral to various biological processes. We previously utilized a protein affinity probe to identify genes damaged by cisplatin, revealing that it inflicts substantial damage on protein kinase and protein phosphatase genes. In this study, we investigated cisplatin-induced alterations in the global proteome and phosphoproteome of A549 cells. Employing Fe-IMAC beads and tyrosine phosphorylation enrichment antibodies, we identified 6944 protein groups and 18,274 phosphorylation sites on 4915 proteins across three biological replicates of both cisplatin-treated A549 cells and control cells. Among these, 730 tyrosine phosphorylation sites were identified-marking the most substantial discovery of such sites in A549 cells following cisplatin treatment. Bioinformatics analysis indicated that the proteins exhibiting significant phosphorylation level changes predominantly involved in RNA processing, modification, transcription, translation, and the spliceosome. This suggests that cisplatin-induced damage to protein kinases and phosphatases may disrupt the normal function of these proteins, consequently impairing DNA replication, RNA translation, and shearing, ultimately culminating in tumor cell death. Moreover, we cross-referenced our proteomic data with our previously obtained cisplatin-damaged genes, observing that the majority of down-regulated proteins derived from cisplatin-induced gene damage. The data are available on ProteomeXchange under the identifier PXD053902., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

CDK5 plays a crucial role in maintaining normal central nervous system (CNS) development and synaptic function, while microglia are the primary immune cells present in the CNS and play vital physiological roles in CNS development, immune surveillance, and regulation of synaptic plasticity. Despite this, our understanding of both the substrate proteins and functional mechanisms of CDK5 in microglia remains limited. To address this, we utilized CRISPR-Cas9 knockout of Cdk5 in BV2 cells and conducted quantitative phosphoproteomics analysis to systematically screen potential CDK5 substrates in microglia. Our findings identified 335 phosphorylation sites on 234 proteins as potential CDK5 substrates in microglia based on the reported sequence motif. Through in vitro kinase assay and intracellular inhibition and knockout of CDK5 experiments, we confirmed that ER proteins MTDH (protein LYRIC) and Calnexin are novel substrate proteins of CDK5. Moreover, we demonstrated for the first time a critical mechanism for regulating protein synthesis in microglia, that the phosphorylation of S565 site on MTDH, a key protein mediating cell growth, by CDK5 inhibits protein synthesis. Our data provide valuable insights for the discovery of new substrate proteins of CDK5 and the in-depth investigation of the function and mechanism of CDK5 in microglia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in nontumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the EGF receptor inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (pyruvate kinase muscle isozyme, phosphoglycerate mutase 1) and two uncharacterized (glutathione S-transferase Pi 1, glutamate dehydrogenase 1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous reexpression of wild-type, phosphomutant, or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization, and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance., Competing Interests: Competing interests statement:A.T. is a consultant for NovoNordisk Holdings, Inc. and receives funding support from BioHybrid Solutions. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals; is listed as an inventor on a patent (WO2019232403A1, Weill Cornell Medicine) for combination therapy for PI3K-associated disease or disorder, and the identification of therapeutic interventions to improve response to PI3K inhibitors for cancer treatment; is a co-founder and shareholder in Faeth Therapeutics; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. T.M.Y. is a co-founder of DeStroke. F.M.W. is a consultant and on the scientific advisory board for Crossbow Therapeutics and for Aethon Therapeutics.

Increasing evidence suggests that mutations in the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may enhance viral replication by modulating the assembly process. However, the mechanisms governing the selective packaging of viral genomic RNA by the N protein, along with the assembly and budding processes, remain poorly understood. Utilizing a virus-like particles (VLPs) system, we have identified that the C-terminal domain (CTD) of the N protein is essential for its interaction with the membrane (M) protein during budding, crucial for binding and packaging genomic RNA. Notably, the isolated CTD lacks M protein interaction capacity and budding ability. Yet, upon fusion with the N-terminal domain (NTD) or the linker region (LKR), the resulting NTD/CTD and LKR/CTD acquire RNA-dependent interactions with the M protein and acquire budding capabilities. Furthermore, the presence of the C-tail is vital for efficient genomic RNA encapsidation by the N protein, possibly regulated by interactions with the M protein. Remarkably, the NTD of the N protein appears dispensable for virus particle assembly, offering the virus adaptive advantages. The emergence of N* (NΔN209) in the SARS-CoV-2 B.1.1 lineage corroborates our findings and hints at the potential evolution of a more streamlined N protein by the SARS-CoV-2 virus to facilitate the assembly process. Comparable observations have been noted with the N proteins of SARS-CoV and HCoV-OC43 viruses. In essence, these findings propose that β-coronaviruses may augment their replication by fine-tuning the assembly process.IMPORTANCEAs a highly transmissible zoonotic virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve. Adaptive mutations in the nucleocapsid (N) protein highlight the critical role of N protein-based assembly in the virus's replication and evolutionary dynamics. However, the precise molecular mechanisms of N protein-mediated viral assembly remain inadequately understood. Our study elucidates the intricate interactions between the N protein, membrane (M) protein, and genomic RNA, revealing a C-terminal domain (CTD)-based assembly mechanism common among β-coronaviruses. The appearance of the N* variant within the SARS-CoV-2 B.1.1 lineage supports our conclusion that the N-terminal domain (NTD) of the N protein is not essential for viral assembly. This work not only enhances our understanding of coronavirus assembly mechanisms but also provides new insights for developing antiviral drugs targeting these conserved processes., Competing Interests: The authors declare no conflict of interest.

Interferon (IFN)-γ inducible protein 16 (IFI16), a key DNA sensor, triggers downstream STING-dependent type I interferon (IFN-I) production and antiviral immunity. However, how the IFI16-STING signaling pathway is regulated by EMCV infection is still not well elucidated. In this study, we investigated the interaction between IFI16 and EMCV. Results indicated EMCV infection suppressed IFI16 expression in A549 cells. This study reveals that IFI16 plays an active role in combating EMCV. Screening viral proteins in conjunction with IFI16, we found that the EMCV VP2 protein hinders the antiviral response mediated by IFI16 by causing degradation of the IFI16 protein via the caspase-dependent apoptosis pathway. Our study communicates the antiviral role of the IFI16-STING pathway during EMCV infection. Importantly, this study unveils the novel mechanism by which VP2 counteracts the innate immune signaling activated by foreign DNA., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Despite the importance of radiation therapy as a nonsurgical treatment for non-small cell lung cancer (NSCLC), radiation resistance has always been a concern because of poor patient response and outcomes. Therefore, it is crucial to identify novel targets to increase the effectiveness of radiotherapy and investigate the mechanisms underlying radioresistance. Previously, we demonstrated that Spindlin 1 (SPIN1) was related to tumour initiation and progression. In this study, we found that SPIN1 expression was higher in NSCLC tissues and cell lines than in the corresponding controls. SPIN1 overexpression in NSCLC patients was closely correlated with disease progression and poor prognosis. Functionally, SPIN1 depletion inhibited cell proliferation, decreased the percentage of cells in the G2/M phase and suppressed cell migration and invasion. Moreover, SPIN1 knockdown decreased the clonogenic capacity, impaired double-strand break (DSB) repair and increased NSCLC radiosensitivity. Mechanistically, forkhead box M1 (FOXM1) was identified as a key downstream effector of SPIN1 in NSCLC cells. Furthermore, SPIN1 was found to facilitate MDM2-mediated FOXO3a ubiquitination and degradation, leading to FOXM1 upregulation. Moreover, restoration of FOXM1 expression markedly abolished the inhibitory effects and increased radiosensitivity induced by SPIN1 depletion. These results indicate that the SPIN1-MDM2-FOXO3a/FOXM1 signalling axis is essential for NSCLC progression and radioresistance and could serve as a therapeutic target for increasing radiotherapy efficacy., Competing Interests: Competing interests The authors declare no competing interests. Ethical approval All the human tissues used in our work were approved by the ethic committee of the First Affiliated Hospital of Nanchang University (No: 2022-3-028). And the animal experiments were also performed under the permission of ethic committee of the First Affiliated Hospital of Nanchang University. This study conformed to the provisions of the Declaration of Helsinki., (© 2024. The Author(s).)

Pediatric burn injuries are a leading cause of morbidity with infections being the most common acute complication. Thermal injuries elicit a heightened cytokine response while suppressing immune function; however, the mechanisms leading to this dysfunction are still unknown. Our aim was to identify extracellular proteins and circulating phosphoprotein expression in the plasma after burn injury to predict the development of nosocomial infection (NI). Plasma was collected within 72 hours after injury from 64 pediatric burn subjects; of these, 18 went on to develop an NI. Extracellular damage-associated molecular proteins, FAS(APO), and protein kinase b (AKT) signaling phosphoproteins were analyzed. Subjects who went on to develop an NI had elevated high-mobility group box 1, heat shock protein 90 (HSP90), and FAS expression than those who did not develop an NI after injury (NoNI). Concurrently, phosphorylated (p-)AKT and mammalian target of rapamycin (p-mTOR) were elevated in those subjects who went on to develop an NI. Quadratic discriminant analysis revealed distinct differential profiles between NI and NoNI burn subjects using HSP90, FAS, and p-mTOR. The area under the receiver-operator characteristic curves displayed significant ability to distinguish between these 2 burn subject cohorts. These findings provide insight into predicting the signaling proteins involved in the development of NI in pediatric burn patients. Further, these proteins show promise as a diagnostic tool for pediatric burn patients at risk of developing infection while additional investigation may lead to potential therapeutics to prevent NI., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control serum phosphate levels by downregulating the renal Na-phosphate transporter NPT2A, thereby decreasing phosphate absorption and augmenting urinary excretion. This mechanism requires NHERF1, a PDZ scaffold protein, and is governed by the regulator of G protein signaling-14 (RGS14), which harbors a carboxy-terminal PDZ ligand that binds NHERF1. RGS14 is part of a triad of structurally related RGS proteins that includes RGS12 and RGS10. Like RGS14, RGS12 contains a class 1 PDZ ligand. However, unlike RGS14, the larger RGS12 contains an upstream PDZ-binding domain. The studies outlined here examined and characterized the binding of RGS12 with NHERF1 and NPT2A and its function on hormone-regulated phosphate transport. Immunoblotting experiments revealed RGS12 C-terminal PDZ ligand binding to NHERF1. Further structural analysis disclosed that NPT2A engaged full-length RGS12 and the upstream fragment containing the PDZ domain. Neither the downstream RGS12 portion nor RGS14 interacted with NPT2A. PTH and FGF23 profoundly inhibited phosphate uptake in opossum kidney proximal tubule cells. Transfection with human RGS14, or human RGS12, abolished hormone-sensitive phosphate transport as reported for human proximal tubule cells. RGS12 inhibitory activity resides in the downstream region and is comparable to RGS14. The carboxy-terminal RGS12(667-1447) splice variant is prominently expressed in the kidney and may contribute to regulating hormone-sensitive phosphate transport., Competing Interests: Declaration of competing interest The authors avow that the described work has not been published previously in any form. The manuscript and results are not under consideration for publication elsewhere. All authors approved by and tacitly or explicitly by the authorities where the work was performed. If accepted, the article will not be published elsewhere in any form, in English or in any other language, including electronically without the written consent of the copyright-holder., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with an unmet clinical need, but its epigenetic regulation remains largely undefined. By performing multiomic profiling, we recently revealed distinct super-enhancer (SE) patterns in different subtypes of breast cancer and identified a number of TNBC-specific SEs that drive oncogene expression. One of these SEs, TCOF1 SE, was discovered to play an important oncogenic role in TNBC. However, the molecular mechanisms by which TCOF1 SE promotes the expression of the TCOF1 gene remain to be elucidated. Here, by using combinatorial approaches of DNA pull-down assay, bioinformatics analysis and functional studies, we identified FOSL1 as a key transcription factor that binds to TCOF1 SE and drives its overexpression. shRNA-mediated depletion of FOSL1 results in significant downregulation of TCOF1 mRNA and protein levels. Using a dual-luciferase reporter assay and ChIP-qPCR, we showed that binding of FOSL1 to TCOF1 SE promotes the transcription of TCOF1 in TNBC cells. Importantly, our data demonstrated that overexpression of FOSL1 drives the activation of TCOF1 SE. Lastly, depletion of FOSL1 inhibits tumor spheroid growth and stemness properties of TNBC cells. Taken together, these findings uncover the key epigenetic role of FOSL1 and highlight the potential of targeting the FOSL1-TCOF1 axis for TNBC treatment., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: SARS-CoV-2, a highly contagious coronavirus, is responsible for the global pandemic of COVID-19 in 2019. Currently, it remains uncertain whether SARS-CoV-2 possesses oncogenic or oncolytic potential in influencing tumor progression. Therefore, it is important to evaluate the clinical and functional role of SARS-CoV-2 on tumor progression., Methods: Here, we integrated bioinformatic analysis of COVID-19 RNA-seq data from the GEO database and performed functional studies to explore the regulatory role of SARS-CoV-2 in solid tumor progression, including lung, colon, kidney and liver cancer., Results: Our results demonstrate that infection with SARS-CoV-2 is associated with a decreased expression of genes associated with cancer proliferation and metastasis in lung tissues from patients diagnosed with COVID-19. Several cancer proliferation or metastasis related genes were frequently downregulated in SARS-CoV-2 infected intestinal organoids and human colon carcinoma cells. In vivo and in vitro studies revealed that SARS-CoV-2 nucleocapsid (N) protein inhibits colon and kidney tumor growth and metastasis through the N-terminal (NTD) and the C-terminal domain (CTD). The molecular mechanism indicates that the N protein of SARS-CoV-2 interacts with YBX1, resulting in the recruitment of PKM mRNA into stress granules mediated by G3BP1. This process ultimately destabilizes PKM expression and suppresses glycolysis., Conclusion: Our study reveals a new function of SARS-CoV-2 nucleocapsid protein on tumor progression., (© 2024. The Author(s).)

Background: The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes-associated protein, 5SA [active] mutant), but persistent overexpression of proliferation-inducing genes could lead to hypertrophy and arrhythmia, whereas off-target expression in fibroblasts and macrophages could increase fibrosis and inflammation., Methods and Results: Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte-specific modified mRNA translation system ( YAP5SA CM-SMRTs or GFP CM-SMRTs, respectively). YAP5SA-cardiomyocyte specificity was confirmed via in vitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced- pluripotent stem cells and in human umbilical-vein endothelial cells, and the regenerative potency of YAP5SA CM-SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced-pluripotent stem cells-cardiomyocytes, YAP was abundantly expressed for 3 days after YAP5SA CM-SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near-background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after GFP CM-SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced-pluripotent stem cells-cardiac fibroblasts and human umbilical-vein endothelial cells on day 1 after GFP CM-SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in YAP5SA CM-SMRTs-treated mice than in vehicle-treated control animals, and YAP5SA CM-SMRTs appeared to promote cardiomyocyte proliferation., Conclusions: The CM-SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

Mechanical and biochemical cues intricately activate Yes-associated protein (YAP), which is pivotal for the cellular responses to these stimuli. Recent findings reveal an unexplored role of YAP in influencing the apoptotic process. It has been shown that, on soft matrices, YAP is recruited to small adhesions, phosphorylated at Y357, and translocated into the nucleus triggering apoptosis. Interestingly, YAP Y357 phosphorylation is significantly reduced in larger mature focal adhesions on stiff matrices. Building upon these novel insights, we have developed a stochastic model to delve deeper into the complex dynamics of YAP phosphorylation within integrin adhesions. Our findings emphasize several key points: firstly, increasing the cytosolic diffusion rate of YAP correlates with higher levels of phosphorylated YAP (pYAP); secondly, increasing the number of binding sites and distributing them across the membrane surface, mimicking smaller adhesions, leads to higher pYAP levels, particularly at lower diffusion rates. Moreover, we show that the binding and release rate of YAP to adhesions as well as adhesion lifetimes significantly influence the size effect of adhesion-induced YAP phosphorylation. The results highlight the complex and dynamic interplay between adhesion lifetime, the rate of pYAP unbinding from adhesions, and dephosphorylation rates, collectively shaping overall pYAP levels. In summary, our work advances the understanding of YAP mechanotransduction and opens avenues for experimental validation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aluminum (Al) toxicity adversely impacts soybean (Glycine max) growth in acidic soil. Reversible protein phosphorylation plays an important role in adapting to adverse environmental conditions by regulating multiple physiological processes including signal transduction, energy coupling and metabolism adjustment in higher plant. This study aimed to reveal the Al-responsive phosphoproteins to understand their putative function and involvement in the regulation of Al resistance in soybean root. We used immobilized metal affinity chromatography to enrich the key phosphoproteins from soybean root apices at 0, 4, or 24 h Al exposure. These phosphoproteins were detected using liquid chromatography-tandem mass spectrometry measurement, verified by parallel reaction monitoring (PRM), and functionally characterized via overexpression in soybean hairy roots. A total of 638 and 686 phosphoproteins were identified as differentially enriched between the 4-h and 0-h, and the 24-h and 0-h Al treatment comparison groups, respectively. Typically, the phosphoproteins involved in biological processes including cell wall modification, and RNA and protein metabolic regulation displayed patterns of decreasing enrichment (clusters 3, 5 and 6), however, the phosphoproteins involved in the transport and metabolic processes of various substrates, and signal transduction pathways showed increased enrichment after 24 h of Al treatment. The enrichment of phosphoproteins in organelle organization bottomed after 4 h of Al treatment (cluster 1). Next, we selected 26 phosphoproteins from the phosphoproteomic profiles, assessed their enrichment status using PRM, and detected enrichment patterns similar to those observed via phosphoproteomic analysis. Among them, 15 phosphoproteins were found to reduce the accumulation of Al and callose in Al-stressed soybean root apices when their corresponding genes were individually overexpressed in soybean hairy roots. In summary, the findings of this study facilitated a comprehensive understanding of the protein phosphorylation events involved in Al resistance responses and revealed some critical phosphoproteins that enhance Al resistance in soybean roots., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and has been related to more than 7 million deaths globally since 2019. The association of high levels of IL-6 with severe cases led to the early evaluation of the anti-IL6 inhibitor tocilizumab as a potential treatment, which unfortunately failed to improve survival in many trials. Moreover, little is known about the development of COVID-19 sequelae, and biomarkers are needed to understand and anticipate these processes. Because extracellular vesicles (EVs) play an important role in viral infection and immune response, they could potentially serve as predictive and prognostic biomarkers. We isolated EVs from 39 patients with severe COVID-19, from which 29 received tocilizumab and 10 were considered controls. Blood samples, which were collected at hospitalisation before treatment, at Day 7, and Day 15 during follow-up, were assessed by immunoblot for longitudinal expression of spike (S) and nucleocapsid (N) proteins. Dynamic expression was calculated and compared with clinicopathological and experimental variables. Expression of EV S was validated by immunogold and imaging flow-cytometry, revealing an enrichment in CD9+ EVs. As a result, decreasing expression of EV viral proteins was observed in patients treated with tocilizumab. Moreover, higher increase in EV S was observed in patients with lower antibody response, hyperfibrinogenemia, lower respiratory function, higher blood pressure and shorter outcomes. These findings lay the foundation for future studies characterizing the role of EVs in multiorgan assessment and identifying biomarkers in patients with severe COVID-19 and possible long COVID., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments., (© 2024. The Author(s).)

Hurler-Scheie syndrome (MPS IH/S), also known as mucopolysaccharidosis type I-H/S (MPS IH/S), is a lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase (IDUA) leading to the accumulation of glycosaminoglycans (GAGs) in various tissues, resulting in a wide range of symptoms affecting different organ systems. Postgenomic omics technologies offer the promise to understand the changes in proteome, phosphoproteome, and phosphorylation-based signaling in MPS IH/S. Accordingly, we report here a large dataset and the proteomic and phosphoproteomic analyses of fibroblasts derived from patients with MPS IH/S ( n = 8) and healthy individuals ( n = 8). We found that protein levels of key lysosomal enzymes such as cathepsin D, prosaposin, arylsulfatases (arylsulfatase A and arylsulfatase B), and IDUA were downregulated. We identified 16,693 unique phosphopeptides, corresponding to 4,605 proteins, in patients with MPS IH/S. We found that proteins related to the cell cycle, mitotic spindle assembly, apoptosis, and cytoskeletal organization were differentially phosphorylated in MPS IH/S. We identified 12 kinases that were differentially phosphorylated, including hyperphosphorylation of cyclin-dependent kinases 1 and 2, hypophosphorylation of myosin light chain kinase, and calcium/calmodulin-dependent protein kinases. Taken together, the findings of the present study indicate significant alterations in proteins involved in cytoskeletal changes, cellular dysfunction, and apoptosis. These new observations significantly contribute to the current understanding of the pathophysiology of MPS IH/S specifically, and the molecular mechanisms involved in the storage of GAGs in MPS more generally. Further translational clinical omics studies are called for to pave the way for diagnostics and therapeutics innovation for patients with MPS IH/S.

Background: Breast cancer is a malignancy with a generally poor prognosis. With the advancement of molecular research, we have gained deeper insights into the cellular processes that drive breast cancer development. However, the precise mechanisms remain elusive., Results: Based on the CPTAC database, we found that NEDD9 expression is up-regulated in breast cancer tissues and is associated with poor prognosis in breast cancer patients. Functional experiments showed that NEDD9 promotes tumor growth and metastasis both in vitro and in vivo. Overexpression of NEDD9 disrupts mammary epithelial acinus formation and triggers epithelial-mesenchymal transition in breast cancer cells, effects that are reversed upon NEDD9 gene silencing. Mechanistically, NEDD9 upregulates its expression by inhibiting HDAC4 activity, leading to enhanced H3K9 acetylation of the NEDD9 gene promoter and activation of the FAK/NF-κB signaling pathway. Furthermore, NEDD9 overexpression promotes IL-6 secretion, which further drives breast cancer progression. Notably, NEDD9 activation fosters the pro-tumoral M2 macrophage polarization in the tumor microenvironment. NEDD9 stimulates IL-6 secretion, polarizes monocytes towards an M2-like phenotype, and enhances BC cell invasiveness., Conclusions: These findings suggest that NEDD9 upregulation plays a pivotal role in breast cancer metastasis and macrophage M2 polarization via the FAK/NF-κB signaling axis. Targeting NEDD9 may offer a promising therapeutic approach for breast cancer treatment., Competing Interests: Declaration of competing interest The authors confirm that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Protein phosphorylation is a crucial regulatory mechanism in cell signaling, acting as a molecular switch that modulates protein function. Catalyzed by protein kinases and reversed by phosphoprotein phosphatases, it is essential in both normal physiological and pathological states. Recent advances have uncovered a vast and intricate landscape of protein phosphorylation that include histidine phosphorylation and more unconventional events, such as pyrophosphorylation and polyphosphorylation. Many questions remain about the true size of the phosphoproteome and, more importantly, its site-specific functional relevance. The involvement of unconventional actors such as pseudokinases and pseudophosphatases adds further complexity to be resolved. This review explores recent discoveries and ongoing challenges, highlighting the need for continued research to fully elucidate the roles and regulation of protein phosphorylation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SF3B1 mutations are recurrent in chronic lymphocytic leukemia (CLL), particularly enriched in clinically aggressive stereotyped subset #2. To investigate their impact, we conducted RNA-sequencing of 18 SF3B1 MUT and 17 SF3B1 WT subset #2 cases and identified 80 significant alternative splicing events (ASEs). Notable ASEs concerned exon inclusion in the non-canonical BAF (ncBAF) chromatin remodeling complex subunit, BRD9, and splice variants in eight additional ncBAF complex interactors. Long-read RNA-sequencing confirmed the presence of splice variants, and extended analysis of 139 CLL cases corroborated their association with SF3B1 mutations. Overexpression of SF3B1 K700E induced exon inclusion in BRD9, resulting in a novel splice isoform with an alternative C-terminus. Protein interactome analysis of the BRD9 splice isoform revealed augmented ncBAF complex interaction, while exhibiting decreased binding of auxiliary proteins, including SPEN, BRCA2, and CHD9. Additionally, integrative multi-omics analysis identified a ncBAF complex-bound gene quartet on chromosome 1 with higher expression levels and more accessible chromatin in SF3B1 MUT CLL. Finally, Cancer Dependency Map analysis and BRD9 inhibition displayed BRD9 dependency and sensitivity in cell lines and primary CLL cells. In conclusion, spliceosome dysregulation caused by SF3B1 mutations leads to multiple ASEs and an altered ncBAF complex interactome, highlighting a novel pathobiological mechanism in SF3B1 MUT CLL., (© 2024. The Author(s).)

Background: Hypertrophic cardiomyopathy (HCM) is a common genetic heart disease. Women with HCM tend to have a later onset but more severe disease course. However, the underlying pathobiological mechanisms for these differences remain unknown., Methods: Myectomy samples from 97 patients (53 males/44 females) with symptomatic obstructive HCM and 23 control cardiac tissues were included in this study. RNA-sequencing was performed on all samples. Mass spectrometry-based proteomics and phosphoproteomics was performed on a representative subset of samples., Results: The transcriptome, proteome, and phosphoproteome was similar between sexes and did not separate on PCA plotting. Overall, there were 482 differentially expressed genes (DEGs) between control females and control males while there were only 53 DEGs between HCM females and HCM males. There were 1983 DEGs between HCM females and control females compared to 1064 DEGs between HCM males and control males. Additionally, there was increased transcriptional downregulation of hypertrophy pathways in HCM females and in HCM males. HCM females had 119 differentially expressed proteins compared to control females while HCM males only had 27 compared to control males. Finally, the phosphoproteome showed females had 341 differentially phosphorylated proteins (DPPs) compared to controls while males only had 184. Interestingly, there was hypophosphorylation and inactivation of hypertrophy pathways in females but hyperphosphorylation and activation in males., Conclusion: There are subtle, but biologically relevant differences in the multi-omics profile of HCM. This study provides the most comprehensive atlas of sex-specific differences in the transcriptome, proteome, and phosphoproteome present at the time of surgical myectomy for obstructive HCM., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: The nucleocapsid protein (N protein) is the most abundant protein in SARS-CoV-2. Viral RNA and this protein are bound by electrostatic forces, forming cytoplasmic helical structures known as nucleocapsids. Subsequently, these nucleocapsids interact with the membrane (M) protein, facilitating virus budding into early secretory compartments., Scope of Review: Exploring the role of the N protein in the SARS-CoV-2 life cycle, pathogenesis, post-sequelae consequences, and interaction with host immunity has enhanced our understanding of its function and potential strategies for preventing SARS-CoV-2 infection., Major Conclusion: This review provides an overview of the N protein's involvement in SARS-CoV-2 infectivity, highlighting its crucial role in the virus-host protein interaction and immune system modulation, which in turn influences viral spread., General Significance: Understanding these aspects identifies the N protein as a promising target for developing effective antiviral treatments and vaccines against SARS-CoV-2., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aim: Lack of adequate mechanical strength and progressive shrinkage over time remain challenges in scaffold-free microtissue-based dental pulp regeneration. Surface collagen cross-linking holds the promise to enhance the mechanical stability of microtissue constructs and trigger biological regulations. In this study, we proposed a novel strategy for surface preconditioning microtissues using a natural collagen cross-linker, proanthocyanidin (PA). We evaluated its effects on cell viability, tissue integrity, and biomineralization of dental pulp stem cell (DPSCs)-derived 3D cell spheroids., Methodology: Microtissue and macrotissue spheroids were fabricated from DPSCs and incubated with PA solution for surface collagen cross-linking. Microtissue viability was examined by live/dead staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with transverse dimension change monitored. Microtissue surface stiffness was measured by an atomic force microscope (AFM). PA-preconditioned microtissues and macrotissues were cultured under basal or osteogenic conditions. Immunofluorescence staining of PA-preconditioned microtissues was performed to detect dentin sialophosphoprotein (DSPP) and F-actin expressions. PA-preconditioned macrotissues were subjected to histological analysis, including haematoxylin-eosin (HE), alizarin red, and Masson trichrome staining. Immunohistochemistry staining was used to detect alkaline phosphatase (ALP) and dentin matrix acidic phosphoprotein 1 (DMP-1) expressions., Results: PA preconditioning had no adverse effects on microtissue spheroid viability and increased surface stiffness. It reduced dimensional shrinkage for over 7 days in microtissues and induced a larger transverse-section area in the macrotissue. PA preconditioning enhanced collagen formation, mineralized nodule formation, and elevated ALP and DMP-1 expressions in macrotissues. Additionally, PA preconditioning induced higher F-actin and DSPP expression in microtissues, while inhibition of F-actin activity by cytochalasin B attenuated PA-induced dimensional change and DSPP upregulation., Conclusion: PA surface preconditioning of DPSCs spheroids demonstrates excellent biocompatibility while effectively enhancing tissue structure stability and promoting biomineralization. This strategy strengthens tissue integrity in DPSC-derived spheroids and amplifies osteogenic differentiation potential, advancing scaffold-free tissue engineering applications in regenerative dentistry., (© 2024 The Author(s). International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

Hexavalent chromium (Cr(VI)) causes testicular damage and reduces testosterone secretion. Testosterone synthesis relies on cholesterol as a raw material, and its availability can be affected by lipophagy. However, the role of lipophagy in Cr(VI)-induced testicular damage and reduced testosterone secretion remains unclear. In this study, we investigated the effect of Cr(VI) on lipid metabolism and lipophagy in the testes of ICR mice. Forty mice were randomly divided into four groups and exposed to different doses of Cr(VI) (0, 75, 100, 125 mg/kg) for thirty days. Cr(VI) increased the rate of sperm abnormalities, decreased testosterone level, and decreased the levels of testosterone synthesis-related proteins, namely steroidogenic acute regulatory (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) proteins. Through metabolomic analysis, Oil Red O staining, and biochemical indicator (triglyceride and total cholesterol) analysis, Cr(VI) was found to disrupt testicular lipid metabolism. Further investigation revealed that Cr(VI) inhibited the AMP-activated protein kinase (AMPK)/sterol regulatory element-binding protein 1 (SREBP1) pathway, elevated levels of the autophagy-related proteins microtubule-associated protein 1 light chain 3B (LC3B) and sequestosome 1 (SQSTM1)/P62 and lipophagy-related proteins Rab7 and Rab10, while increasing colocalization of LC3B and Perilipin2. These findings suggest that Cr(VI) exposure leads to abnormal lipid metabolism in the testes by suppressing the AMPK/SREBP1 pathway and disrupting lipophagy, ultimately reducing testosterone level and inducing testicular damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Steroidogenesis occurs locally in peripheral tissues and via adrenal and gonadal glands' biosynthesis. The C2C12 mouse myoblast cell line and rat skeletal muscles harbor a local steroidogenesis pathway for glucocorticoids, and corticosterone is biosynthesized from skeletal muscle cells. However, Cyp11a1 and StAR protein expressions are not observed in C2C12 cells or rat muscular tissues. In this context, this study investigated the relationship between DNA methylation and key steroidogenic genes. Bioinformatics analysis of methylated DNA immune precipitation showed that C2C12 myoblasts and myotubes did not have remarkable DNA methylated regions in the gene-body of Cyp11a1. However, a highly methylated region in the CpG island was detected in the intronic enhancer of Ad4BP/SF-1, known as the transcriptional factor for steroidogenic genes. After C2C12 myoblasts treatment with 5-aza-2-deoxycytidine, the gene expressions of Ad4BP/SF-1, Cyp11a1, and StAR were significantly time- and concentration-dependent upregulated. To clarify the contribution of Ad4BP/SF-1 on Cyp11a1 and StAR transcripts, we silenced Ad4BP/SF-1 during the 5-aza-2-deoxycytidine treatment in C2C12 myoblasts, resulting in significant suppression of both Cyp11a1 and StAR. Additionally, pregnenolone levels in the supernatants of C2C12 cells were enhanced by 5-aza-2-deoxycytidine treatment, whereas pregnenolone production by C2C12 myoblasts was significantly suppressed by Ad4BP/SF-1 knockdown. These results indicate that DNA methylation of Ad4BP/SF-1 might be involved in the downregulation of steroidogenic genes, such as Cyp11a1 and StAR in C2C12 myoblasts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Dentin phosphophoryn (DPP) an extracellular matrix protein activates Wnt signaling in DPSCs (dental pulp stem cells). Wnt/β catenin signaling is essential for tooth development but the role of DPP-mediated Wnt5a signaling in odontogenesis is not well understood. Wnt5a is typically considered as a non-canonical Wnt ligand that elicits intracellular signals through association with a specific cohort of receptors and co-receptors in a cell and context-dependent manner. In this study, DPP facilitated the interaction of Wnt5a with Frizzled 5 and LRP6 to induce nuclear translocation of β-catenin. β-catenin has several nuclear binding partners that promote the activation of Wnt target genes responsible for odontogenic differentiation. Interestingly, steady increase in the expression of Vangl2 receptor suggest planar cell polarity signaling during odontogenic differentiation. In vitro observations were further strengthened by the low expression levels of Wnt5a and β-catenin in the teeth of DSPP KO mice which exhibit impaired odontoblast differentiation and defective dentin mineralization. Together, this study suggests that the DPP-mediated Wnt5a signaling could be exploited as a therapeutic approach for the differentiation of dental pulp stem cells into functional odontoblasts and dentin regeneration., (© 2024. The Author(s).)

Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (PhosTAC)-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, we show that, counter to our current understanding, many PP1 regulators are also substrates, that the number of residues between regulator PP1-binding and phosphosites vary significantly, and that PP1 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

Genetic variants involving steroidogenic acute regulatory protein cause lipoid congenital adrenal hyperplasia, which is characterized by impaired steroidogenesis in the adrenal glands and gonads. Functional assessment of variant STAR proteins is necessary for an accurate genetic diagnosis. Ideally, steroidogenic cells should be used to assess the functionality of STAR proteins, but the presence of endogenous STARs in steroidogenic cells precludes such a method. Here, we generated Star-edited cells from steroidogenic Y1 mouse adrenocortical tumor cells by genome editing. Star-edited Y1 cells exhibited very low but measurable cAMP-dependent pregnenolone production. Furthermore, stimulation of the cAMP pathway for 2 weeks resulted in the formation of lipid droplets in the cytoplasm of Star-edited Y1 cells, which resembled the histology of the adrenal glands of patients with lipoid congenital adrenal hyperplasia. The steroidogenic defect of Star-edited Y1 cells can be restored by transient overexpression of mouse Star. We found that human STAR can also restore defective steroidogenesis in Star-edited Y1 cells, and we were able to construct a novel in vitro system to evaluate human STAR variants. Collectively, we established Star-edited Y1 cells that retain the steroidogenic pathway downstream of the Star protein. Star-edited Y1 cells recapitulate the functional and morphological changes of lipoid congenital adrenal hyperplasia and can be used to evaluate the functionality of human STAR variants.

The concentration of many transcription factors exhibits high cell-to-cell variability due to differences in synthesis, degradation, and cell size. Whether the functions of these factors are robust to fluctuations in concentration, and how this may be achieved, is poorly understood. Across two independent panels of breast cancer cells, we show that the average whole cell concentration of YAP decreases as a function of cell area. However, the nuclear concentration distribution remains constant across cells grouped by size, across a 4-8 fold size range, implying unperturbed nuclear translocation despite the falling cell wide concentration. Both the whole cell and nuclear concentration was higher in cells with more DNA and CycA/PCNA expression suggesting periodic synthesis of YAP across the cell cycle offsets dilution due to cell growth and/or cell spreading. The cell area - YAP scaling relationship extended to melanoma and RPE cells. Integrative analysis of imaging and phospho-proteomic data showed the average nuclear YAP concentration across cell lines was predicted by differences in RAS/MAPK signalling, focal adhesion maturation, and nuclear transport processes. Validating the idea that RAS/MAPK and cell cycle regulate YAP translocation, chemical inhibition of MEK or CDK4/6 increased the average nuclear YAP concentration. Together, this study provides an example case, where cytoplasmic dilution of a protein, for example through cell growth, does not limit a cognate cellular function. Here, that same proteins translocation into the nucleus.

There are only a few effective molecular targeted agents for advanced unresectable or recurrent advanced gastric cancer (AGC), which has a poor prognosis with a median survival time of less than 14 months. Focusing on phosphorylation signaling in cancer cells, we have been developing deep phosphoproteome analysis from minute endoscopic biopsy specimens frozen within 20 s of collection. Phosphoproteomic analysis of 127 fresh-frozen endoscopic biopsy samples from untreated patients with AGC revealed three subtypes reflecting different cellular signaling statuses. Subsequent serial biopsy analysis has revealed the dynamic mesenchymal transitions within cancer cells, along with the concomitant rewiring of the kinome network, ultimately resulting in the conversion to the epithelial-mesenchymal transition (EMT) subtype throughout treatment. We present our investigation of intracellular signaling related to the EMT in gastric cancer and propose therapeutic approaches targeting AXL. This study also provides a wealth of resources for the future development of treatments and biomarkers for AGC., Competing Interests: Declaration of interests T.T. and J.A. are co-founders of Proteobiologics Co., Ltd., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Nipah virus (NiV) is a highly pathogenic paramyxovirus causing frequently lethal encephalitis in humans. The NiV genome is encapsidated by the nucleocapsid (N) protein. RNA synthesis is mediated by the viral RNA-dependent RNA polymerase (RdRP), consisting of the polymerase (L) protein complexed with the homo-tetrameric phosphoprotein (P). The advance of the polymerase along its template requires iterative dissolution and reformation of transient interactions between P and N protomers in a highly regulated process that remains poorly understood. This study applied functional and biochemical NiV polymerase assays to the problem. We mapped three distinct protein interfaces on the C-terminal P-X domain (P-XD), which form a triangular prism and engage L, the C-terminal N tail, and the globular N core, respectively. Transcomplementation assays using NiV L and N-tail binding-deficient mutants revealed that only one XD of a P tetramer binds to L, whereas three must be available for N-binding for efficient polymerase activity. The dissolution of the N-tail complex with P-XD was coordinated by a transient interaction between N-core and the α-1/2 face of this XD but not unoccupied XDs of the P tetramer, creating a timer for coordinated polymerase advance., Importance: Mononegaviruses comprise major human pathogens such as the Ebola virus, rabies virus, respiratory syncytial virus, measles virus, and Nipah virus (NiV). For replication and transcription, their polymerase complexes must negotiate a protein-encapsidated RNA genome, which requires the highly coordinated continuous formation and resolution of protein-protein interfaces as the polymerase advances along the template. The viral P protein assumes a central role in this process, but the molecular mechanism of ensuring polymerase mobility is poorly understood. Studying NiV polymerase complexes, we applied functional and biochemical assays to map three distinct interfaces in the NiV P XD and identified transient interactions between XD and the nucleocapsid core as instrumental in coordinating polymerase advance. These results define a conserved molecular principle regulating paramyxovirus polymerase dynamics and illuminate a promising druggable target for the structure-guided development of broad-spectrum polymerase inhibitors., Competing Interests: The authors declare no conflict of interest.

Background: Immune checkpoint inhibitor (ICIs)-based therapies are the standard of care treatment for patients with metastatic melanoma (MM). The stimulator of interferon genes (STING) signaling pathway is critical in controlling immune responses to ICIs. Interferon (IFN)-γ-inducible protein 16 (IFI16) is a cytosolic DNA sensor that activates the STING signaling pathway. The link between IFI16 and STING signaling pathway on IFN-γ stimulation and the connection to ICIs response remains not completely understood., Methods: Deconvolution analyses were performed using the TCGA-SKCM, GSE91061, and PRJEB23709 public RNA sequencing (RNA-seq) data sets that contained RNA-seq for patients with MM. Functional assays combined with cytokine arrays were performed using MM cell lines to validate in silico data. Multiplex immunofluorescence was performed on untreated or pretreatment tumor samples from patients with MM., Results: Deconvolution analysis showed that high- IFI16 levels in melanoma cells were associated with a good prognosis in patients with MM and positively correlated with M1-macrophage infiltration. Functional assays using MM cell lines demonstrated that IFI16 is a key molecule to sense cytosolic DNA and activate STING and nuclear factor kappa B (NF-κB) signaling pathways, independent of cyclic GMP-AMP synthase or absent in melanoma 2, on IFN-γ stimulation. IFI16 knockdown significantly decreased CXCL10 and ICAM1 secretion. EZH2 inhibitor reversed the repressive epigenetic control on IFI16 to promote STING and NF-κB signaling pathways on IFN-γ stimulation. Increased IFI16, ICAM1, and CXCL10 levels in tumor samples from patients with MM were positively correlated with M1-macrophage infiltration and a significantly better response to ICIs., Conclusions: This study identifies IFI16 as a key sensor during IFN-γ stimulation associated with ICI response, and it proposes the epigenetic EZH2 inhibitor as an alternative treatment strategy to overcome ICI resistance in patients with MM., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The ability of the attaching and effacing pathogens enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium to overcome colonisation resistance is reliant on a type 3 secretion system used to intimately attach to the colonic epithelium. This crucial virulence factor is encoded on a pathogenicity island known as the Locus of Enterocyte Effacement (LEE) but its expression is regulated by several core-genome encoded transcription factors. Here, we unveil that the core transcription factor PdhR, traditionally known as a regulator of central metabolism in response to cellular pyruvate levels, is a key activator of the LEE. Through genetic and molecular analyses, we demonstrate that PdhR directly binds to a specific motif within the LEE master regulatory region, thus activating type 3 secretion directly and enhancing host cell adhesion. Deletion of pdhR in EHEC significantly impacted the transcription of hundreds of genes, with pathogenesis and protein secretion emerging as the most affected functional categories. Furthermore, in vivo studies using C. rodentium, a murine model for EHEC infection, revealed that PdhR is essential for effective host colonization and maximal LEE expression within the host. Our findings provide new insights into the complex regulatory networks governing bacterial pathogenesis. This research highlights the intricate relationship between virulence and metabolic processes in attaching and effacing pathogens, demonstrating how core transcriptional regulators can be co-opted to control virulence factor expression in tandem with the cell's essential metabolic circuitry., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wale et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A decrease in environmental temperature represents a challenge to the cardiovascular system of ectotherms. To gain insight into the cellular changes that occur during cold exposure and cold acclimation we characterized the cardiac phosphoproteome and proteome of zebrafish following 24 h or 1 week exposure to 20°C from 27°C; or at multiple points during 6 weeks of acclimation to 20°C from 27°C. Our results indicate that cold exposure causes an increase in mitogen-activated protein kinase signalling, the activation of stretch-sensitive pathways, cellular remodelling via ubiquitin-dependent pathways and changes to the phosphorylation state of proteins that regulate myofilament structure and function including desmin and troponin T. Cold acclimation (2-6 weeks) led to a decrease in multiple components of the electron transport chain through time, but an increase in proteins for lipid transport, lipid metabolism, the incorporation of polyunsaturated fatty acids into membranes and protein turnover. For example, there was an increase in the levels of apolipoprotein C, prostaglandin reductase-3 and surfeit locus protein 4, involved in lipid transport, lipid metabolism and lipid membrane remodelling. Gill opercular movements suggest that oxygen utilization during cold acclimation is reduced. Neither the amount of food consumed relative to body mass nor body condition was affected by acclimation. These results suggest that while oxygen uptake was reduced, energy homeostasis was maintained. This study highlights that the response of zebrafish to a decrease in temperature is dynamic through time and that investment in the proteomic response increases with the duration of exposure., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Female crabs enter a stage of rapid ovarian development after mating, and cholesterol is a substrate for steroid hormone synthesis. Therefore, in this experiment, an 8-week feeding trial was conducted to investigate the effects of mating treatments (mated crab and unmated crab) and three dietary cholesterol levels (0·09 %, 0·79 % and 1·40 %) on ovarian development, cholesterol metabolism and steroid hormones metabolism of adult female swimming crab ( Portunus trituberculatus ). The results indicated that crabs fed the diet with 0·79 % cholesterol significantly increased gonadosomatic index (GSI) and vitellogenin (VTG) content than other treatments in the same mating status. Moreover, mated crabs had markedly increased GSI and VTG content in the ovary and hepatopancreas than unmated crabs. The histological observation found that exogenous vitellogenic oocytes appeared in the mated crabs, while previtellogenic oocytes and endogenous vitellogenic oocytes were the primary oocytes in unmated crabs. The transmission electron microscopy analysis showed that when fed diet with 0·79 % cholesterol, the unmated crabs contained more rough endoplasmic reticulum and mated crabs had higher yolk content than other treatments. Furthermore, mating treatment and dietary 0·79 % cholesterol level both promoted cholesterol deposition by up-regulation of the mRNA and protein expression levels of class B scavenger receptors 1 (Srb1), while stimulating the secretion of steroid hormones by up-regulation of the mRNA and protein expression of steroidogenic acute regulatory protein (Star). Overall, the present results indicated that mating behaviour plays a leading role in promoting ovarian development, and dietary 0·79 % cholesterol level can further promote ovarian development after mating.

Considering the properties of myo-inositol (MI) and D-chiro-inositol (DCI), which are well known in polycystic ovary syndrome therapy, and the limitations of adult granulosa cell tumor (AGCT) treatment, especially for androgen-secreting tumors, we studied the role of MI and DCI in the androgen-rich environment of AGCTs. For this purpose, we analyzed the mRNA expression of steroidogenic genes and the secretion of progesterone (P4) and 17β-estradiol (E2) in an unstimulated and/or dihydrotestosterone (DHT)-stimulated environment under MI and DCI influence. Thus, we used the HGrC1 and KGN cell lines as in vitro models of healthy and cancerous granulosa cells. We found that DHT, the most potent androgen, increased E2 secretion and steroidogenic acute regulatory protein ( StAR ) and cytochrome P450 side-chain cleavage gene ( CYP11A1 ) mRNA expression without affecting 450 aromatase ( CYP19A1 ) in AGCTs. However, after the MI and DCI treatment of KGN cells, both compounds strongly reduced StAR and CYP11A1 expression. Interestingly, in DHT-stimulated KGN cells, only DCI alone and its cotreatment with MI reduced both CYP11A1 mRNA and E2 secretion. These findings suggest that CYP11A1 is responsible for the antiestrogenic effect of DCI in the androgen-rich environment of AGCTs. Therefore, MI and DCI could be used as effective agents in the adjuvant treatment of AGCT, but further detailed studies are needed.

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.

Neurotrophic receptor tyrosine kinases (TrkA, TrkB, TrkC), despite their homology, contribute to the clinical heterogeneity of the childhood cancer neuroblastoma. TrkA expression is associated with low-stage disease and is often seen with spontaneous tumour regression. Conversely, TrkB is present in unfavourable neuroblastomas that often harbour amplification of the MYCN oncogene. The role of TrkC is less clearly defined, although some studies suggest its association with a favourable outcome. Understanding the differences in activity of Trk receptors that drive divergent clinical phenotypes as well as the influence of MYCN amplification on downstream Trk receptor signalling remains poorly understood. Here, we present a comprehensive label-free mass spectrometry-based total proteomics and phosphoproteomics dataset (432 raw files with FragPipe search outputs; available on PRIDE with accession number PXD054441) where we identified and quantified 4,907 proteins, 16,744 phosphosites and 5,084 phosphoproteins, derived from NGF/BDNF/NT-3 treated TrkA/B/C-overexpressing neuroblastoma cells with differential MYCN status. Analysing our dataset offers valuable insights into TrkA/B/C receptor signalling in neuroblastoma and its modulation by MYCN status; and holds potential for advancing therapeutic strategies in this challenging childhood cancer., (© 2024. The Author(s).)

The steroidogenic acute regulatory (STAR) protein is an essential cholesterol transporter that shuttles cholesterol from the outer to the inner mitochondrial membrane in the major steroidogenic endocrine organs. It is a key player in the acute regulation of steroid hormone biosynthesis in response to tropic hormone stimulation. Its discovery 30 years ago sparked immediate interest in understanding how STAR action is controlled. Since increased STAR gene expression is a classic feature of the acute regulation of steroidogenesis, a special emphasis was placed on defining the transcriptional regulatory mechanisms that underlie its rapid induction in response to tropic hormone stimulation. These mechanisms include the effects of enhancers, the regulation of chromatin accessibility, the impact of epigenetic factors, and the role of transcription factors. Over the past three decades, understanding the transcription factors that regulate STAR gene expression has been the subject of more than 170 independent scientific publications, making it one of, and if not the best, studied genes in the steroidogenic pathway. This intense research effort has led to the identification of dozens of transcription factors and their related binding sites in STAR 5' flanking (promoter) sequences across multiple species. STAR gene transcription appears to be complex in that a large number of transcription factors have been proposed to interact with either isolated or overlapping regulatory sequences that are tightly clustered over a relatively short promoter region upstream of the STAR transcription start site. Many of these transcription factors appear to work in unique combinatorial codes and are impacted by diverse hormonal and intracellular signaling pathways. This review provides a retrospective overview of the transcription factors proposed to regulate both basal and acute (hormonal) STAR gene expression, and how insights in this area have evolved over the past 30 years.

Background: Breast cancer ranks among the most prevalent tumor types worldwide. Copy number amplification of chromosome 8q24 is frequently detected in breast cancer. ZNF623 is a relatively unexplored gene mapped to 8q24. Here, we explore the expression profile, prognostic significance, and biological action of ZNF623 in breast carcinogenesis., Methods: To evaluate the mRNA expression pattern and prognostic relevance of ZNF623 across different cancer types, we conducted bioinformatic analyses. The expression of the gene was suppressed using ZNF623 shRNAs/siRNAs and augmented through transfection with plasmids containing ZNF623 cDNA. Cell viability assay, clonogenic assay, and transwell migration assay were utilized to assess the proliferation, viability, and invasion capacity of breast cancer cell lines. Luciferase reporter assay served as a pivotal tool to ascertain the transcriptional activity of ZNF623. IP-MS and co-IP were employed to validate that ZNF623 interacted with CtBP1. ChIP analysis and ChIP-qPCR were conducted to assess the genes targeted by ZNF623/CtBP1 complex. Flow cytometry was conducted to evaluate the phosphorylation status of p65., Results: ZNF623 expression was notably elevated in breast cancer (BC). Prognostic analysis indicated higher expression of ZNF623 indicated worse survival. Functional experiments discovered that the upregulation of ZNF623 significantly enhanced both the proliferative and migratory capacities of breast cancer cells. Luciferase reporter assay indicated that ZNF623 was a transcription repressor. Immunoprecipitation coupled mass spectrometry analysis revealed a physical association between ZNF623 and CtBP1 in the interaction group. The conjoint analysis of ChIP-seq and TCGA DEG analysis revealed that the ZNF623/CtBP1 complex repressed a series of genes, such as negative regulation of the NF-kappaB signaling pathway. Flow cytometry analysis discovered that knockdown of ZNF623 decreased the phosphorylation level of p65, indicating that ZNF623 could regulate the activity of the NF-κB pathway., Conclusion: ZNF623 predicts poor prognosis of BC and enhances breast cancer growth and metastasis. By recruiting CtBP1, ZNF623 could suppress NF-κB inhibitors, including COMMD1, NFKBIL1, PYCARD, and BRMS1, expression from the transcription level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Nipah virus (NiV), a member of the Paramyxoviridae family, is notorious for its high fatality rate in humans. The RNA polymerase machinery of NiV, comprising the large protein L and the phosphoprotein P, is essential for viral replication. This study presents the 2.9-Å cryo-electron microscopy structure of the NiV L-P complex, shedding light on its assembly and functionality. The structure not only demonstrates the molecular details of the conserved N-terminal domain, RNA-dependent RNA polymerase (RdRp), and GDP polyribonucleotidyltransferase of the L protein, but also the intact central oligomerization domain and the C-terminal X domain of the P protein. The P protein interacts extensively with the L protein, forming an antiparallel β-sheet among the P protomers and with the fingers subdomain of RdRp. The flexible linker domain of one P promoter extends its contact with the fingers subdomain to reach near the nascent RNA exit, highlighting the distinct characteristic of the NiV L-P interface. This distinctive tetrameric organization of the P protein and its interaction with the L protein provide crucial molecular insights into the replication and transcription mechanisms of NiV polymerase, ultimately contributing to the development of effective treatments and preventive measures against this Paramyxoviridae family deadly pathogen., (© 2024. The Author(s).)

In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast., (© 2024. The Author(s).)

The dormancy of cancer stem cells is a major factor leading to drug resistance and a high rate of late recurrence and mortality in estrogen receptor-positive (ER+) breast cancer. Previously, we demonstrated that a stiffer matrix induces tumor cell dormancy and drug resistance, whereas a softened matrix promotes tumor cells to exhibit a stem cell state with high proliferation and migration. In this study, we present a comprehensive analysis of the proteome and phosphoproteome in response to gradient changes in matrix stiffness, elucidating the mechanisms behind cell dormancy-induced drug resistance. Overall, we found that antiapoptotic and membrane transport processes may be involved in the mechanical force-induced dormancy resistance of ER+ breast cancer cells. Our research provides new insights from a holistic proteomic and phosphoproteomic perspective, underscoring the significant role of mechanical forces stemming from the stiffness of the surrounding extracellular matrix as a critical regulatory factor in the tumor microenvironment.

Atypical enteropathogenic Escherichia coli (aEPEC) is a significant cause of diarrhea in low- and middle-income countries. Certain aEPEC strains, including the Brazilian representative strain of serotype O51:H40 called aEPEC 1711-4, can use flagella to attach to, invade, and persist in T84 and Caco-2 intestinal cells. It can also translocate from the gut to extraintestinal sites in a rat model. Although various aspects of the virulence of this strain were studied and the requirement of a type III secretion system for the efficiency of the invasion process was demonstrated, the expression of the locus of enterocyte effacement (LEE) genes during the invasion and intracellular persistence remains unclear. To address this question, the expression of flagella and the different LEE operons was evaluated during kinetic experiments of the interaction of aEPEC 1711-4 with enterocytes in vitro . The genome of the strain was also sequenced. The results showed that flagella expression remained unchanged, but the expression of eae and escJ increased during the early interaction and invasion of aEPEC 1711-4 into Caco-2 cells, and there was no change 24 h post-infection during the persistence period. The number of actin accumulation foci formed on HeLa cells also increased during the 6-h analysis. No known gene related to the invasion process was identified in the genome of aEPEC 1711-4, which was shown to belong to the global EPEC lineage 10. These findings suggest that the LEE components and the intimate adherence promoted by intimin are necessary for the invasion and persistence of aEPEC 1711-4, but the detailed mechanism needs further study.IMPORTANCEAtypical enteropathogenic Escherichia coli (aEPEC) is a major cause of diarrhea, especially in low- and middle-income countries, like Brazil. However, due to the genome heterogeneity of each clonal group, it is difficult to comprehend the pathogenicity of this strain fully. Among aEPEC strains, 1711-4 can invade eukaryotic cells in vitro , cross the gut barrier, and reach extraintestinal sites in animal models. By studying how different known aEPEC virulence factors are expressed during the invasion process, we can gain insight into the commonalities of this phenotype among other aEPEC strains. This will help in developing preventive measures to control infections caused by invasive strains. No known virulence-encoding genes linked to the invasion process were found. Nevertheless, additional studies are still necessary to evaluate the role of other factors in this phenotype., Competing Interests: The authors declare no conflict of interest.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing., Competing Interests: Declaration of interests O.A.-W. is a founder and scientific advisor of Codify Therapeutics, holds equity in this company, and receives research funding from this company. O.A.-W. has served as a consultant for Foundation Medicine Inc., Merck, Prelude Therapeutics, Amphista Therapeutics, MagnetBio, and Janssen, and is on the Scientific Advisory Board of Envisagenics Inc., Harmonic Discovery Inc., and Pfizer Boulder; O.A.-W. has received prior research funding from H3B Biomedicine, Nurix Therapeutics, Minovia Therapeutics, and LOXO Oncology unrelated to the current manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Splicing modulation by a small compound offers therapeutic potential for diseases caused by splicing abnormality. However, only a few classes of compounds that can modulate splicing have been identified. We previously identified BAY61-3606, a multiple kinase inhibitor, as a compound that relaxes the splicing fidelity at the 3' splice site recognition. We have also reported the synthesis of derivatives of BAY61-3606. In this study, we tested those compounds for their splicing modulation capabilities and identified two contrasting compounds. These compounds were further investigated for their effects on the whole transcriptome, and analysis of changes in transcription and splicing revealed that the highly active derivative in the splicing reporter assay also showed significantly higher activity in modulating the splicing of endogenously expressed genes. Particularly, cassette exon inclusion was highly upregulated by this compound, and clustering analysis revealed that these effects resembled those in splicing factor 3b subunit 1 (SF3B1) K700E mutant cells but contrasted with those of the splicing inhibitor H3B-8800. Additionally, a group of serine/arginine-rich (SR) protein genes was identified as representatively affected, likely via modulation of poison exon inclusion. This finding could guide further analysis of the mode of action of these compounds on splicing, which could be valuable for developing drugs for diseases associated with splicing abnormalities., (© 2024 John Wiley & Sons Ltd.)

Introduction. Enteropathogenic Escherichia coli (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins. Gap Statement. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in ler expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation. Aim. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Methodology. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity. Results. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression. Conclusion. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.

Purpose: Unfortunately, treatment of patients with uveal melanoma (UM) with metastatic disease is limited. Twenty percent of patients with UM harbor a mutation in the splicing factor gene SF3B1, suggesting that aberrant spliceosome function plays a vital role in tumorigenesis. Splicing inhibitors exploit the preferential sensitivity of spliceosome-compromised leukemic cells to these compounds., Methods: We studied the effect of the splicing inhibitor E7107 using two UM cell lines and ex vivo cultured SF3B1- and BAP1-mutated primary UM tumor slices. These UM cell lines and ex vivo tumor slices were exposed for 24 hours to different concentrations of E7107. Tumor slices were stained with hematoxylin and eosin (H&E) and incubated with BAP1, MelanA, MIB-1, and caspase-3 antisera., Results: The E7107-exposed UM cell lines exhibited decreased cell viability and increased apoptosis, with the greatest effect on SF3B1-mutated UM cells. A similar effect on UM tumor slices was observed upon exposure to E7107. Additionally, RNA was isolated for differential isoform expression analysis. No significant difference in isoform usage was found genome-wide. However, specific genes were differentially expressed after E7107 treatment in the SF3B1-mutated samples. Moreover, E7107 had the greatest effect on intron retention., Conclusions: This study indicates/suggests that mutated SF3B1 UM cells are more sensitive to the splicing inhibitor E7107 than wild-type SF3B1 UM cells.

The concomitant activation of both the YAP1 co-transcription factor and RAS GTPases is a hallmark of several aggressive cancers, though the intricacies of their relationship and implications for oncogenesis are still poorly understood. This review has presented a cooperative model where YAP1 and RAS are not independently acting oncogenes but rather interdependently acting ones, with each fulfilling an essential role within the oncogenic process. YAP1 is responsible for initiating the expression of key proteins that contribute to various cancer traits. However, these proteins must often be transported into the cytoplasm to exert their effects. We suggest that oncogenic RAS actually facilitates this transport, enabling the phosphorylation and subsequent activation of the nuclear transporter XPO1 (aka Exportin1). This mechanism is particularly crucial for anti-apoptotic proteins. Instead of being sequestered within the nucleus in an ineffective state, these proteins are rather shuttled into the cytoplasm. Within the cytoplasm, they can effectively inhibit apoptosis, undermining by these means the efficacy of chemotherapeutic agents designed to induce cell death in cancer cells. Therefore, a clearer understanding of the oncogenic partnership between RAS and YAP1 will likely provide new insights into the molecular underpinnings of cancer and highlight as well potential targets for therapeutic interventions designed to disrupt this pernicious interaction., (© 2024. The Author(s).)