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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).)

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).)

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).)

Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) play fundamental roles in stem/progenitor cell expansion during homeostasis, and their dysregulation often leads to tissue overgrowth. Here, we show that YAP activation is sufficient to overcome the restriction of global protein synthesis induced by serum starvation, enabling cells to sustain proliferation and survival despite an unfavorable environment. Mechanistically, YAP/TAZ selectively promoted the mTORC1-dependent translation of mRNAs containing 5' terminal oligopyrimidine (5'TOP) motifs, ultimately increasing the cellular polysome content. Interestingly, DNA damage-inducible transcript 4 (DDIT4), a negative regulator of mTORC1, was upregulated by serum starvation but repressed by YAP/TAZ. DDIT4 was sufficient to suppress the translation and transformative potential of uveal melanoma cells, which are often serum unresponsive due to G protein mutations. Our findings reveal a vital role for protein synthesis as a key modality of YAP/TAZ-induced oncogenic transformation and indicate the potential for targeting mTORC1 or translation to treat YAP/TAZ-driven malignancies., (© 2024. The Author(s).)

The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young, active transposons during germline development in male mice 1 . piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript, resulting in DNA methylation through SPOCD1 (refs. 2-5 ). Transposon methylation requires great precision: every copy needs to be methylated but off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 interacts directly with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds to H3K4me3-K9me3 (ref. 6 ). The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1 copies, but not old ones, are marked by H3K4me3, H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process. The first authentication is the recruitment of SPIN1-SPOCD1 to the young LINE1 promoter, and the second is MIWI2 engagement with the nascent transcript. In summary, independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation., (© 2024. The Author(s).)

The RNA helicase DDX21 is vital for ribosome biogenesis and is upregulated in CRC, but the mechanism by which DDX21 is dysregulated and by which DDX21 promotes tumorigenesis in CRC remains poorly understood. Here, we showed that DDX21 is a direct transcriptional target gene of β-catenin and mediates the protumorigenic function of β-catenin in CRC. DDX21 expression is correlated with the expression and activity of β-catenin, and high DDX21 expression is associated with a poor prognosis in CRC patients. Loss of DDX21 leads to cytoplasmic translocation and decreased transcriptional activity of YAP and suppresses the proliferation and migration of CRC cells, which can be partially rescued by YAP reactivation. Importantly, by using translation elongation inhibitors and DNA intercalators, we showed that ribosomal stress upregulates DDX21 expression and induces the downregulation of LATS and the activation of YAP, probably through the ZAKα-MKK4/7-JNK axis. Overall, our study revealed the transcriptional activation mechanism of DDX21 in CRC and the activation of YAP in the ribosomal stress response, indicating the potential of combination therapy involving the induction of ribosomal stress and YAP inhibition., (© 2024. The Author(s).)

Phosphorylation is a key post-translational modification regulating protein function and biological outcomes. However, the phosphorylation dynamics orchestrating mammalian oocyte development remains poorly understood. In the present study, we apply high-resolution mass spectrometry-based phosphoproteomics to obtain the first global in vivo quantification of mouse oocyte phosphorylation. Of more than 8000 phosphosites, 75% significantly oscillate and 64% exhibit marked upregulation during meiotic maturation, indicative of the dominant regulatory role. Moreover, we identify numerous novel phosphosites on oocyte proteins and a few highly conserved phosphosites in oocytes from different species. Through functional perturbations, we demonstrate that phosphorylation status of specific sites participates in modulating critical events including metabolism, translation, and RNA processing during meiosis. Finally, we combine inhibitor screening and enzyme-substrate network prediction to discover previously unexplored kinases and phosphatases that are essential for oocyte maturation. In sum, our data define landscape of the oocyte phosphoproteome, enabling in-depth mechanistic insights into developmental control of germ cells., (© 2024. The Author(s).)

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known. Here, we use Saccharomyces cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, the resulting tyrosine phosphorylation is biologically spurious. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3500 proteins. The number of spurious pY sites generated correlates strongly with decreased growth, and we predict over 1000 pY events to be deleterious. However, we also find that many of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with tyrosine kinases. Our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve., (© 2024. The Author(s).)

Yes-associated protein (YAP) and its homolog, transcriptional coactivator with PDZ-binding motif (TAZ), are the main transcriptional downstream effectors of the Hippo pathway. Decreased Hippo pathway activity leads to nuclear translocation of YAP/TAZ where they interact with TEAD transcription factors to induce target gene expression. Unrestrained YAP/TAZ activity can lead to excessive growth and tumor formation in a short time, underscoring the evolutionary need for tight control of these two transcriptional coactivators. Here, we report that the AP-1 component JUN acts as specific repressor of YAP/TAZ at joint target sites to decrease YAP/TAZ activity. This function of JUN is independent of its heterodimeric AP-1 partner FOS and the canonical AP-1 function. Since expression of JUN is itself induced by YAP/TAZ, our work identifies a JUN-dependent negative feedback loop that buffers YAP/TAZ activity at joint genomic sites. This negative feedback loop gets disrupted in liver cancer to unlock the full oncogenic potential of YAP/TAZ. Our results thus demonstrate an additional layer of control for the interplay of YAP/TAZ and AP-1., (© 2024. The Author(s).)

This study aimed to investigate the expression of SF3B1 in non-small cell lung cancer, and its clinical significance, biological function, and molecular mechanisms. SF3B1 mRNA and protein levels were elevated in both lung squamous cell carcinoma and lung adenocarcinoma (LUAD) tissues based on TCGA data and immunohistochemistry. Notably, high SF3B1 expression in LUAD was significantly associated with increased lymph node metastasis. Functional experiments involving SF3B1 knockdown and overexpression demonstrated that SF3B1 facilitated the proliferation, invasion, and migration of LUAD cells. Additionally, the SF3B1 inhibitor pladienolide-B attenuated the aggressive behavior of LUAD cells both in vitro and in vivo. RNA sequencing analysis indicated that differentially expressed genes in the SF3B1 knockdown and SF3B1 inhibitor groups were enriched in ferroptosis-related pathways compared to their respective control groups. The antiferroptotic role of SF3B1 in LUAD cells was validated by detecting glutathione depletion, lipid peroxidation, and observing morphological changes using transmission electron microscopy. This process was confirmed to be independent of apoptosis and autophagy, as evidenced by the effects of the ferroptosis inducer erastin, the apoptosis inhibitor Z-VAD-FMK, and the autophagy inhibitor 3-methyladenine. Rescue experiments indicated that the antiferroptotic role of SF3B1 in LUAD is partially mediated by upregulating the expression of SLC7A11., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CDK1 has been known to be the sole cyclin-dependent kinase (CDK) partner of cyclin B1 to drive mitotic progression 1 . Here we demonstrate that CDK5 is active during mitosis and is necessary for maintaining mitotic fidelity. CDK5 is an atypical CDK owing to its high expression in post-mitotic neurons and activation by non-cyclin proteins p35 and p39 2 . Here, using independent chemical genetic approaches, we specifically abrogated CDK5 activity during mitosis, and observed mitotic defects, nuclear atypia and substantial alterations in the mitotic phosphoproteome. Notably, cyclin B1 is a mitotic co-factor of CDK5. Computational modelling, comparison with experimentally derived structures of CDK-cyclin complexes and validation with mutational analysis indicate that CDK5-cyclin B1 can form a functional complex. Disruption of the CDK5-cyclin B1 complex phenocopies CDK5 abrogation in mitosis. Together, our results demonstrate that cyclin B1 partners with both CDK5 and CDK1, and CDK5-cyclin B1 functions as a canonical CDK-cyclin complex to ensure mitotic fidelity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Hepatocellular carcinoma (HCC) stands as one of the most aggressively advancing and lethal malignancies. Sorafenib is presently endorsed as a primary therapy for advanced liver cancer, but its resistance presents a formidable challenge. Previous studies have implicated a connection between post-sorafenib discontinuation rebound and the development of drug resistance, yet the underlying mechanism remains elusive. In this study, we discerned that Sorafenib induced a senescent phenotype in HCC cells and caused a cleavage of ubiquitin-binding protein p62. Mechanistic studies establish that truncated p62 drives cellular senescence by promoting proteasome-dependent degradation of 4EBP1. Furthermore, truncated p62 induced specific ubiquitination of 4EBP1. Crucially, virtual drug screening uncovered that dacinostat inhibited cellular senescence by blocking sorafenib-induced p62 cleavage. In summary, our findings imply that truncated p62 from sorafenib cleavage promotes senescence via 4EBP1 degradation. The prevention of p62 cleavage could emerge as a crucial strategy for impeding the sorafenib-induced cellular senescence., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinases, especially of Ret and the FGF receptors. As such, they play distinct and overlapping roles in embryo morphogenesis and are considered to be tumor suppressors in adult life. Genetic experiments in mice have defined in great detail the role of these genes during embryonic development, however their function in adult mice is less clearly established. Here we generate adult-onset, whole body Spry1/2/4 triple knockout mice. Tumor incidence in triple mutant mice is comparable to that of wild type littermates of up to one year of age, indicating that Sprouty loss per se is not sufficient to initiate tumorigenesis. On the other hand, triple knockout mice do not gain weight as they age, show less visceral fat, and have lower plasma glucose levels than wild type littermates, despite showing similar food intake and slightly reduced motor function. They also show alopecia, eyelid inflammation, and mild hyperthyroidism. Finally, triple knockout mice present phosphaturia and hypophosphatemia, suggesting exacerbated signaling downstream of FGF23. In conclusion, triple knockout mice develop a series of endocrine abnormalities but do not show increased tumor incidence., (© 2024. The Author(s).)

Anaplastic thyroid carcinoma (ATC) is a highly aggressive human malignancy without effective treatment. Yes-associated protein (YAP) is a critical effector of the Hippo pathway, which is essential in thyroid carcinogenesis. However, the underlying mechanisms of aberrant YAP expression in ATC are not completely understood. Ubiquitylation-related enzyme siRNA screening identified the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) as a stabilizer of YAP in ATC cells. UBR1 deficiency reduced YAP protein levels and its target gene expression. UBR1 directly interacted with YAP and promoted its monoubiquitylation, competitively suppressing its polyubiquitylation and resulting in extended protein half-life. UBR1 depletion reduced ATC cell proliferation and migration in vitro. Xenograft tumor studies also suggested that UBR1 knockdown suppressed ATC cell growth in vivo. Furthermore, exogenous YAP expression partially reversed the inhibitive effects of UBR1 depletion on ATC cell proliferation and migration. Our studies demonstrated that UBR1 directly interacts with YAP and stabilized it in a monoubiquitylation-dependent manner, consequently promoting ATC tumorigenesis, suggesting that UBR1 might be a potentially therapeutic target for ATC treatment., (© 2024. The Author(s).)

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins 1-3 . The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour 3,4 . The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors 5-9 . Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P 2 , phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain., (© 2024. The Author(s).)

Targeting intracellular inhibiting proteins has been revealed to be a promising strategy to improve CD8 + T cell anti-tumor efficacy. Here, we are focusing on intracellular inhibiting proteins specific to TCR signaling: DOK1 and DOK2 expressed in T cells. We hypothesized that depletion of intracellular inhibition checkpoint DOK1 and DOK2 could improve CD8 + T-cell based cancer therapies. To evaluate the role of DOK1 and DOK2 depletion in physiology and effector function of CD8 + T lymphocytes and in cancer progression, we established a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) in WT and Dok1/Dok2 DKO (double KO) mice. We showed that both DOK1 and DOK2 depletion in CD8 + T cells after an in vitro pre-stimulation induced a higher percentage of effector memory T cells as well as an up regulation of TCR signaling cascade- induced by CD3 mAbs, including the increased levels of pAKT and pERK, two major phosphoproteins involved in T cell functions. Interestingly, this improved TCR signaling was not observed in naïve CD8 + T cells. Despite this enhanced TCR signaling essentially shown upon stimulation via CD3 mAbs, pre-stimulated Dok1/Dok2 DKO CD8 + T cells did not show any increase in their activation or cytotoxic capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8 + T cells. Indeed, our results allow us to conclude that DOK1 and DOK2 have an inhibitory role following long term T cell stimulations., (© 2024. The Author(s).)

Tissue repair, immune defence and cancer progression rely on a vital cellular decision between quiescence and proliferation 1,2 . Mammalian cells proliferate by triggering a positive feedback mechanism 3,4 . The transcription factor E2F activates cyclin-dependent kinase 2 (CDK2), which in turn phosphorylates and inactivates the E2F inhibitor protein retinoblastoma (Rb). This action further increases E2F activity to express genes needed for proliferation. Given that positive feedback can inadvertently amplify small signals, understanding how cells keep this positive feedback in check remains a puzzle. Here we measured E2F and CDK2 signal changes in single cells and found that the positive feedback mechanism engages only late in G1 phase. Cells spend variable and often extended times in a reversible state of intermediate E2F activity before committing to proliferate. This intermediate E2F activity is proportional to the amount of phosphorylation of a conserved T373 residue in Rb that is mediated by CDK2 or CDK4/CDK6. Such T373-phosphorylated Rb remains bound on chromatin but dissociates from it once Rb is hyperphosphorylated at many sites, which fully activates E2F. The preferential initial phosphorylation of T373 can be explained by its relatively slower rate of dephosphorylation. Together, our study identifies a primed state of intermediate E2F activation whereby cells sense external and internal signals and decide whether to reverse and exit to quiescence or trigger the positive feedback mechanism that initiates cell proliferation., (© 2024. The Author(s).)

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth 1 . Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome 1-3 . How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood 4-7 . Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution., (© 2024. The Author(s).)

RNA-binding proteins (RBPs)-RNA networks have contributed to cancer development. Circular RNAs (circRNAs) are considered as protein recruiters; nevertheless, the patterns of circRNA-protein interactions in colorectal cancer (CRC) are still lacking. Processing bodies (PBs) formed through liquid-liquid phase separation (LLPS) are membrane-less organelles (MLOs) consisting of RBPs and RNA. Previous evidence suggests a connection between PBs dynamics and cancer progression. Despite the increasingly acknowledged crucial role of RBPs and RNA in the accumulation and maintenance of MLOs, there remains a lack of specific research on the interactions between PBs-related RBPs and circRNAs in CRC. Herein, we identify that MEX-3 RNA binding family member A (MEX3A), frequently upregulated in CRC tissues, predicts poorer patient survival. Elevated MEX3A accelerates malignance and inhibits autophagy of CRC cells. Importantly, MEX3A undergoes intrinsically disordered regions (IDRs)-dependent LLPS in the cytoplasm. Specifically, circMPP6 acts as a scaffold to facilitate the interaction between MEX3A and PBs proteins. The MEX3A/circMPP6 complex modulates PBs dynamic and promotes UPF-mediated phosphodiesterase 5A (PDE5A) mRNA degradation, consequently leading to the aggressive properties of CRC cells. Clinically, CRC patients exhibiting high MEX3A expression and low PDE5A expression have the poorest overall survival. Our findings reveal a collaboration between MEX3A and circMPP6 in the regulation of mRNA decay through triggering the PBs aggregation, which provides prognostic markers and/or therapeutic targets for CRC., (© 2024. The Author(s).)

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with K d values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD., (© 2024. The Author(s).)

Eukaryotic initiation factor 4E (eIF4E) is a pivotal protein involved in the regulatory mechanism for global protein synthesis in both physiological and pathological conditions. MicroRNAs (miRNAs) play a significant role in regulating gene expression by targeting mRNA. However, the ability of miRNAs to regulate eIF4E and its phosphorylation remains relatively unknown. In this study, we predicted and experimentally verified targets for miR-483-5p, including eukaryotic translation initiation factor eIF4E and its binding proteins, 4E-BPs, that regulate protein synthesis. Using the Web of Science database, we identified 28 experimentally verified miR-483-5p targets, and by the TargetScan database, we found 1818 predicted mRNA targets, including EIF4E, EIF4EBP1, and EIF4EBP2. We verified that miR-483-5p significantly reduced ERK1 and MKNK1 mRNA levels in HEK293 cells. Furthermore, we discovered that miR-483-5p suppressed EIF4EBP1 and EIF4EBP2, but not EIF4E. Finally, we found that miR-483-5p reduced the level of phosphorylated eIF4E (pSer209eIF4E) but not total eIF4E. In conclusion, our study suggests that miR-483-5p's multi-targeting effect on the ERK1/ MKNK1 axis modulates the phosphorylation state of eIF4E. Unlike siRNA, miRNA can have multiple targets in the pathway, and thereby exploring the role of miR-483-5p in various cancer models may uncover therapeutic options., (© 2024. The Author(s).)

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation 1 . Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases 2 , whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55 3 . Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited 4 . Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP19 5,6 and FAM122A 7 . Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases., (© 2023. The Author(s).)

In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A1 1-4 . Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19 F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function., (© 2023. The Author(s).)

Splicing factor 3B subunit 1 (SF3B1) is the largest component of SF3b protein complex which is involved in the pre-mRNA splicing mechanism. Somatic mutations of SF3B1 were shown to be associated with aberrant splicing, producing abnormal transcripts that drive cancer development and/or prognosis. In this study, we focus on the relationship between SF3B1 and four types of cancer, namely myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) and breast cancer (BC). For this purpose, we identified from the Pubmed library only articles which mentioned SF3B1 in connection with the investigated types of cancer for the period 2007 to 2018 to reveal how the connection has developed over time. We left out all published articles which mentioned SF3B1 in other contexts. We retrieved the target articles and investigated the association between SF3B1 and the mentioned four types of cancer. For this we utilized some of the publicly available databases to retrieve gene/variant/disease information related to SF3B1. We used the outcome to derive and analyze a variety of complex networks that reflect the correlation between the considered diseases and variants associated with SF3B1. The results achieved based on the analyzed articles and reported in this article illustrated that SF3B1 is associated with hematologic malignancies, such as MDS, AML, and CLL more than BC. We found that different gene networks may be required for investigating the impact of mutant splicing factors on cancer development based on the target cancer type. Additionally, based on the literature analyzed in this study, we highlighted and summarized what other researchers have reported as the set of genes and cellular pathways that are affected by aberrant splicing in cancerous cells., (© 2023. The Author(s).)

Protein phosphorylation is one of the most widespread post-translational modifications in biology 1,2 . With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes 3,4 . For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible 3 . Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways., (© 2023. The Author(s).)

Atypical chemokine receptor 3 (ACKR3) has emerged as a key player in various biological processes. Its atypical "intercepting receptor" properties have established ACKR3 as the major regulator in the pathophysiological processes in many diseases. In this study, we investigated the role of ACKR3 activation in promoting colorectal tumorigenesis. We showed that ACKR3 expression levels were significantly increased in human colon cancer tissues, and high levels of ACKR3 predicted the increased severity of cancer. In Villin-ACKR3 transgenic mice with a high expression level of CKR3 in their intestinal epithelial cells, administration of AOM/DSS induced more severe colorectal tumorigenesis than their WT littermates. Cancer cells of Villin-ACKR3 transgenic mice were characterised by the nuclear β-arrestin-1 (β-arr1)-activated perturbation of rRNA biogenesis. In HCT116 cells, cotreatment with CXCL12 and AMD3100 selectively activated ACKR3 and induced nuclear translocation of β-arr1, leading to an interaction of β-arr1 with nucleolar and coiled-body phosphoprotein 1 (NOLC1). NOLC1, as the phosphorylated protein, further interacted with fibrillarin, a conserved nucleolar methyltransferase responsible for ribosomal RNA methylation in the nucleolus, thereby increasing the methylation in histone H2A and promoting rRNA transcription in ribosome biogenesis. In conclusion, ACKR3 promotes colorectal tumorigenesis through the perturbation of rRNA biogenesis by the β-arr1-induced interaction of NOLC1 with fibrillarin., (© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Gastric cancer is one of the most lethal human malignancies in the world. Although great efforts are put in developing novel therapeutic targets, the effective targeting drugs are still limited. Recent studies reveal the abnormality of Hippo/YAP axis play critical role in the oncogenic process of gastric cancer. It is of great importance to demonstrate the regulation of Hippo signaling activity and YAP protein turnover in gastric cancer. Besides, the phosphorylation cascade on YAP function, which has been thoroughly investigated, the ubiquitination of YAP is also important in Hippo signaling status. Here, We utilized the DUB (Deubiquitinase) siRNA library to identify critical DUB for Hippo signaling. We discovered OTUB1 as a critical factor to facilitate gastric cancer cell stemness and progression, which deubiquitinated and stabilized YAP protein. The clinical data analysis implicated OTUB1 was higher expressed in gastric cancer, which correlated with YAP activity and poor survival. OUTB1 interacted with YAP protein via its OTU domain (Ovarian tumor domain) and deubiquitinated YAP at several lysine sites (K90, K280, K343, K494 and K497), which subsequently inhibited YAP degradation. Our study revealed a novel deubiquitinase of Hippo/YAP axis and one possible therapeutic target for YAP-driven gastric cancer., (© 2022. The Author(s).)

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes 1,2 . Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex 1-5 . Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein 6 -is phosphorylated during spliceosome activation 7-10 , but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the B act complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex B act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The water-soluble β-cyclodextrin-curcumin (CDC) is used in pharmaceutical applications and as a natural food colorant. The previous study revealed that curcumin potentially impacted the reproductive system. The present study investigated the possible roles of the CDC in testosterone secretion in Leydig cells and mice. Primary Leydig cells were treated with the CDC to determine their effect on cell proliferation, testosterone levels, the protein and mRNA expression of the transcription factor, and steroidogenic enzymes. Our data showed that CDC stimulated testosterone production via upregulating transcription factor steroidogenic factor-1 (NR5A1), cAMP-response element-binding protein (CREB), and steroidogenic enzymes steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (CYP11A1), 17-alpha-hydroxylase/17,20-lyase (CYP17A1), 3β-/17β-hydroxysteroid dehydrogenase type 1 (3β/17β-HSD, HSD3b1/HSD17b1). CDC could significantly stimulate H89-suppressed StAR and CREB expression but not reverse melatonin-suppressed StAR expression. We further detected the hormonal activity with transgenic yeast, and CDC showed potential androgenic antagonistic activity. Meanwhile, we investigated its aphrodisiac effect on hydrocortisone-induced mice. Exposure to hydrocortisone decreased the mating ability, reproductive organs, and testosterone level and disrupted testicular histology. However, all of these effects were significantly improved by CDC treatment. In conclusion, these results indicated that mechanisms of CDC in stimulating testosterone production involve upregulating the cAMP-PKA pathway., (© 2022. The Author(s).)

Epigenetic alterations have emerged as fundamental players in development and progression of breast cancer (BC). A hypoxic tumour microenvironment regulates the stemness phenotype in breast cancer stem cells (BCSCs). The aim of this study was to investigate Znhit1 and HIF-2α gene expression in breast cancer tissues as well as their relation to CSCs markers LGR5, ALDH1A1 and β-catenin in tissue and serum of BC patients. The present study included 160 females divided into two groups, group I: 80 healthy females served as control group and group II: 80 breast cancer patients. Gene expression of tissue Znhit1 and HIF-2α was determined by qRT-PCR. Tissue and serum ALDH1A1, LGR5 and β-catenin levels were determined by ELISA. We found that gene expression of Znhit1 was significantly downregulated in BC tissues. Moreover, it was significantly negatively correlated with clinical stage and β-catenin levels in BC patients. Regarding HIF-2α, gene expression of HIF-2α was significantly upregulated in BC tissues. Moreover, it was significantly positively correlated with Her-2/neu expression and β-catenin levels in BC patients. Based upon our results, Znhit1 and HIF-2α may serve as novel therapeutic targets for BC therapy. Additionally, each of serum ALDH1A1, LGR5 and β-catenin may play a crucial role in non-invasive detection of BC with a high specificity and sensitivity., (© 2022. The Author(s).)

Cln Three Requiring 9 (CTR9), a scaffold protein of the polymerase-associated factor-1 (PAF1) complex (PAF1c), is primarily localized in the nucleus of cells. Recent studies show that CTR9 plays essential roles in the development of various human cancers and their occurrence; however, its regulatory roles and precise mechanisms in hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the roles of CTR9 using in vitro assays and a xenograft mouse model. We found that CTR9 protein is upregulated in tumor tissues from HCC patients. Knockdown of CTR9 substantially reduced HCC cell proliferation, invasion, and migration, whereas its overexpression promoted these activities. In addition, in vitro results revealed that CTR9 silencing dramatically increased cell cycle regulators, p21 and p27, but markedly decreased matrix metalloproteinases, MMP2 and MMP9, with these outcomes reversed upon CTR9 overexpression. Furthermore, the underlying molecular mechanism suggests that CTR9 promoted the oncogene paternally expressed gene 10 (PEG10) transcription via its promoter region. Finally, the oncogenic roles of CTR9 were confirmed in a xenograft mouse model. This study confirms that CTR9, an oncoprotein that promotes HCC cell proliferation, invasion, and migration, increases tumor growth in a xenograft mouse model. CTR9 could be a novel therapeutic target. Further investigation is warranted to verify CTR9 potential in novel therapies for HCC., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs) 1,2 . However, the principles on which cyclin-CDK complexes organize the temporal order of cell cycle events are contentious 3 . One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events 4-6 . A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity 7,8 . In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events 9,10 . Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs., (© 2022. The Author(s).)

WNT signalling is essential for regulating a diverse range of cellular processes. In skeletal muscle, the WNT pathway plays crucial roles in maintenance of the stem cell pool and myogenic differentiation. Focus is usually directed at examining the function of central components of the WNT pathway, including β-CATENIN and the GSK3β complex and TCF/LEF transcription factors, in tissue homeostasis and cancer. Other core components of the WNT pathway though, are three dishevelled (DVL) proteins: membrane associated proteins that propagate WNT signalling from membrane to nucleus. Here we examined DVL function in human myogenesis and the muscle-related cancer alveolar rhabdomyosarcoma. We demonstrate that DVL1 and DVL3 are necessary for efficient proliferation in human myoblasts and are important for timely myogenic differentiation. DVL1 and DVL3 also contribute to regulation of proliferation in rhabdomyosarcoma. DVL1 or DVL3 must be present in the nucleus to regulate proliferation, but they operate through different protein domains: DVL3 requires the DIX and PDZ domains, while DVL1 does not. Importantly, DVL1 and DVL3 activity is independent of markedly increased translocation of β-CATENIN to the nucleus, normally a hallmark of active canonical WNT signalling., (© 2022. The Author(s).)

Zonula occludens (ZO)-1, a tight-junction protein (TJP), is expressed in dendritic cells (DCs) but not in monocytes, and 27-hydroxycholesterol (27OHChol) drives the differentiation of monocytes into DCs. Because the effects of 27OHChol on ZO-1 are not yet clearly defined, we investigated whether 27OHChol induces expression of the TJP. The treatment of human THP-1 monocytic cells with 27OHChol resulted in the elevated transcript levels of ZO-1 but not of ZO-2 or -3. 27OHChol increased the total amount of ZO-1 protein in the cells as well as its level on the cells surface. Cholesterol, however, did not influence expression of ZO-1. And, the expression of ZO-1 protein was mediated by endoplasmic reticulum (ER)-to-Golgi body transport system. Pharmacological kinase inhibition with LY294002 (a PI3K inhibitor), U0126 (a MEK/ERK inhibitor), or PP2 (a Src family kinase inhibitor) resulted in impaired ZO-1 expression at both transcript and protein levels. Drugs that are reported to suppress DC differentiation also inhibited 27OHChol-mediated expression and the localization of ZO-1, indicating the coincidence of ZO-1 upregulation and DC differentiation. These results suggest that ZO-1 is differentially expressed while monocytes differentiate into DCs in the presence of 27OHChol via pathways in which distinct signaling molecules are involved., (© 2022. The Author(s).)

Autophagic decline is considered a hallmark of ageing. The activity of this intracytoplasmic degradation pathway decreases with age in many tissues and autophagy induction ameliorates ageing in many organisms, including mice. Autophagy is a critical protective pathway in neurons and ageing is the primary risk factor for common neurodegenerative diseases. Here, we describe that autophagosome biogenesis declines with age in mouse brains and that this correlates with increased expression of the SORBS3 gene (encoding vinexin) in older mouse and human brain tissue. We characterise vinexin as a negative regulator of autophagy. SORBS3 knockdown increases F-actin structures, which compete with YAP/TAZ for binding to their negative regulators, angiomotins, in the cytosol. This promotes YAP/TAZ translocation into the nucleus, thereby increasing YAP/TAZ transcriptional activity and autophagy. Our data therefore suggest brain autophagy decreases with age in mammals and that this is likely, in part, mediated by increasing levels of vinexin., (© 2021. The Author(s).)

FXYD1 is a key protein controlling ion channel transport. FXYD1 exerts its function by regulating Na + /K + -ATPase activity, mainly in brain and cardiac tissues. Alterations of the expression level of the FXYD1 protein cause diastolic dysfunction and arrhythmias in heart and decreased neuronal dendritic tree and spine formation in brain. Moreover, FXYD1, a target of MeCP2, plays a crucial role in the pathogenesis of the Rett syndrome, a neurodevelopmental disorder. Thus, the amount of FXYD1 must be strictly controlled in a tissue specific manner and, likely, during development. Epigenetic modifications, particularly DNA methylation, represent the major candidate mechanism that may regulate Fxyd1 expression. In the present study, we performed a comprehensive DNA methylation analysis and mRNA expression level measurement of the two Fxyd1 transcripts, Fxyd1a and Fxyd1b, in brain and heart tissues during mouse development. We found that DNA methylation at Fxyd1a increased during brain development and decreased during heart development along with coherent changes in mRNA expression levels. We also applied ultra-deep methylation analysis to detect cell to cell methylation differences and to identify possible distinct methylation profile (epialleles) distribution between heart and brain and in different developmental stages. Our data indicate that the expression of Fxyd1 transcript isoforms inversely correlates with DNA methylation in developing brain and cardiac tissues suggesting the existence of a temporal-specific epigenetic program. Moreover, we identified a clear remodeling of epiallele profiles which were distinctive for single developmental stage both in brain and heart tissues., (© 2022. The Author(s).)

Numerous emotional and cognitive processes mediated by the hippocampus present differences between sexes and can be markedly influenced by hormonal status in males and females of several species. In rodents, the dorsal hippocampus (dHPC) is known to contribute to the rapid antidepressant actions of the NMDA receptor antagonist ketamine. We and others have demonstrated a greater sensitivity to the fast-acting antidepressant ketamine in female versus male rats that is estrogen- and progesterone-dependent. However, the underlying mechanisms remain unclear. Using an acute low dose (2.5 mg/kg) of ketamine that is behaviorally effective in female but not male rats, a label-free phosphoproteomics approach was employed to identify ketamine-induced changes in signaling pathway activation and phosphoprotein abundance within the dHPC of intact adult male rats and female rats in either diestrus or proestrus. At baseline, males and females showed striking dissimilarities in the dHPC proteome and phosphoproteome related to synaptic signaling and mitochondrial function-differences also strongly influenced by cycle stage in female rats. Notably, phosphoproteins enriched in PKA signaling emerged as being both significantly sex-dependent at baseline and also the primary target of ketamine-induced protein phosphorylation selectively in female rats, regardless of cycle stage. Reduced phosphoprotein abundance within this pathway was observed in males, suggesting bi-directional effects of low-dose ketamine between sexes. These findings present biological sex and hormonal milieu as critical modulators of ketamine's rapid actions within this brain region and provide greater insight into potential translational and post-translational processes underlying sex- and hormone-dependent modulation of ketamine's therapeutic effects., (© 2022. The Author(s).)

Inflammatory bowel diseases, like ulcerative colitis and Crohn's disease are frequently accompanied by colorectal cancers. However, the mechanisms underlying colitis-associated cancers are not fully understood. Src Kinase Associated Phosphoprotein 2 (SKAP2), a substrate of Src family kinases, is highly expressed in macrophages. Here, we examined the effects of SKAP2 on inflammatory responses in a mouse model of tumorigenesis with colitis induced by azoxymethane/dextran sulfate sodium. SKAP2 knockout increased the severity of colitis and tumorigenesis, as well as lipopolysaccharide (LPS) induced acute inflammation. SKAP2 attenuated inflammatory signaling in macrophages induced by uptake of cancer cell-derived exosomes. SKAP2 -/- mice were characterized by the activation of NF-κB signaling and the upregulation and release of cytokines including TNFα, IL-1β, IL-6, CXCL-9/-10/-13, and sICAM1; SKAP2 overexpression attenuated NF-κB activation. Mechanistically, SKAP2 formed a complex with the SHP-1 tyrosine phosphatase via association with the Sirpα transmembrane receptor. SKAP2 also physically associated with the TIR domain of MyD88, TIRAP, and TRAM, adaptors of toll-like receptor 4 (TLR4). SKAP2-mediated recruitment of the Sirpα/SHP-1 complex to TLR4 attenuated inflammatory responses, whereas direct interaction of SKAP2 with SHP-2 decreased SHP-2 activation. SHP-2 is required for efficient NF-κB activation and suppresses the TRAM/TRIF-INFβ pathway; therefore, SKAP2-mediated SHP-2 inhibition affected two signaling axes from TLR4. The present findings indicate that SKAP2 prevents excess inflammation by inhibiting the TLR4-NF-κB pathway, and it activates the TLR4-IFNβ pathway through SHP-1 and SHP-2, thereby suppressing inflammation-mediated tumorigenesis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission 1,2 . Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant 3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection 4 . The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants., (© 2021. The Author(s).)

Colorectal cancer (CRC) is among the top five most common malignant tumors worldwide and has a high mortality rate. Identification of the mechanism of CRC and potential therapeutic targets is critical for improving survival. In the present study, we observed high expression of RAN binding protein 1 (RANBP1) in CRC tissues. Upregulated RANBP1 expression was strongly associated with TNM stages and was an independent risk factor for poor prognosis. In vitro and in vivo functional experiments demonstrated that RANBP1 promoted the proliferation and invasion of CRC cells and inhibited the apoptosis of CRC cells. Low RANBP1 expression reduced the expression levels of hsa-miR-18a, hsa-miR-183, and hsa-miR-106 microRNAs (miRNAs) by inhibiting the nucleoplasmic transport of precursor miRNAs (pre-miRNAs), thereby promoting the accumulation of the latter in the nucleus and reducing the expression of mature miRNAs. Further experiments and bioinformatic analyses demonstrated that RANBP1 promoted the expression of YAP by regulating miRNAs and the Hippo pathway. We also found that YAP acted as a transcriptional cofactor to activate RANBP1 transcription in combination with TEAD4 transcription factor. Thus, RANBP1 further promoted the progression of CRC by forming a positive feedback loop with YAP. Our results revealed the biological role and mechanism of RANBP1 in CRC for the first time, suggesting that RANBP1 can be used as a diagnostic molecule and a potential therapeutic target in CRC., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG's. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM., (© 2022. The Author(s).)

The mouth environment comprises the second most significant microbiome in the body, and its equilibrium is critical in oral health. Secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1), a protein normally produced by the gingival epithelium to mediate its attachment to teeth, was suggested to be bactericidal. Our aim was to further explore the antibacterial potential of human SCPPPQ1 by characterizing its mode of action and identifying its active portions. In silico analysis showed that it has molecular parallels with antimicrobial peptides. Incubation of Porphyromonas gingivalis, a major periodontopathogen, with the full-length protein resulted in decrease in bacterial number, formation of aggregates and membrane disruptions. Analysis of SCPPPQ1-derived peptides indicated that these effects are sustained by specific regions of the molecule. Altogether, these data suggest that human SCPPPQ1 exhibits antibacterial capacity and provide new insight into its mechanism of action., (© 2021. The Author(s).)

Metastasis of bladder cancer is a complex process and has been associated with poor clinical outcomes. However, the mechanisms of bladder cancer metastasis remain largely unknown. The present study found that the long noncoding RNA lnc00892 was significantly downregulated in bladder cancer tissues, with low lnc00892 expression associated with poor prognosis of bladder cancer patients. Lnc00892 significantly inhibited the migration, invasion, and metastasis of bladder cancer cells in vitro and in vivo. In-depth analysis showed that RhoA/C acted downstream of lnc00892 to inhibit bladder cancer metastasis. Mechanistically, lnc00892 reduces nucleolin gene transcription by competitively binding the promoter of nucleolin with c-Jun, thereby inhibiting nucleolin-mediated stabilization of RhoA/RhoC mRNA. Taken together, these findings provide novel insights into understanding the mechanisms of bladder cancer metastasis and suggest that lnc00892 can serve as a potential therapeutic target in patients with invasive bladder cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Dentin phosphophoryn synthesized and processed predominantly by the odontoblasts, functions as both structural and signaling protein. Mechanistic studies revealed that DPP stimulation of DPSCs positively impacted the differentiation of DPSCs into functional odontoblasts. Results show that NF-κB signaling and transcriptional activation of genes involved in odontoblast differentiation were influenced by DPP signaling. Specifically, RelA/p65 subunit of NF-κB was identified as being responsible for the initiation of the differentiation cascade. Confocal imaging demonstrated the nuclear translocation of p65 with DPP stimulation. Moreover, direct binding of nuclear NF-κB p65 subunit to the promoter elements of Runx2, Osx, OCN, MMP1, MMP3, BMP4 and PTX3 were identified by ChIP analysis. Pharmacological inhibition of the NF-κB pathway using TPCA-1, a selective inhibitor of IKK-2 and JSH-23, an inhibitor that prevents nuclear translocation and DNA binding of p65 showed impairment in the differentiation process. Functional studies using Alizarin-Red staining showed robust mineral deposits with DPP stimulation and sparse deposition with defective odontoblast differentiation in the presence of inhibitors. In vivo expression of NF-κB targets such as OSX, OCN, PTX3 and p65 in odontoblasts and dental pulp cells from DSPP null mouse was lower when compared with the wild-type. Overall, the results suggest an important role for DPP-mediated NF-κB activation in the transcriptional regulation of early odontogenic markers that promote differentiation of DPSCs., (© 2021. The Author(s).)

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp -1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a Dspp P19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. Dspp P19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. Dspp P19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp -1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp +/+ and Dspp P19L dentin. The Dspp -1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders., (© 2021. The Author(s).)

Endosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility., (© 2021. The Author(s).)

Expression of β-crystallin B2 (CRYβB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYβB2-induced malignancy and the association of CRYβB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYβB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYβB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYβB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYβB2 and the nucleolar protein, nucleolin. CRYβB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYβB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYβB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYβB2 in primary TNBC correlates with decreased survival. In summary, CRYβB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYβB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The retinoic acid-inducible gene I (RIG-I) receptor senses cytoplasmic viral RNA and activates type I interferons (IFN-I) and downstream antiviral immune responses. How RIG-I binds to viral RNA and how its activation is regulated remains unclear. Here, using IFI16 knockout cells and p204-deficient mice, we demonstrate that the DNA sensor IFI16 enhances IFN-I production to inhibit influenza A virus (IAV) replication. IFI16 positively upregulates RIG-I transcription through direct binding to and recruitment of RNA polymerase II to the RIG-I promoter. IFI16 also binds to influenza viral RNA via its HINa domain and to RIG-I protein with its PYRIN domain, thus promoting IAV-induced K63-linked polyubiquitination and RIG-I activation. Our work demonstrates that IFI16 is a positive regulator of RIG-I signalling during influenza virus infection, highlighting its role in the RIG-I-like-receptor-mediated innate immune response to IAV and other RNA viruses, and suggesting its possible exploitation to modulate the antiviral response.