Post-translational Regulation Research Articles

Identification of tomato F-box proteins functioning in phenylpropanoid metabolism.

Phenylpropanoids, a class of specialized metabolites, play crucial roles in plant growth and stress adaptation and include diverse phenolic compounds such as flavonoids. Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are essential enzymes functioning at the entry points of general phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. In Arabidopsis, PAL and CHS are turned over through ubiquitination-dependent proteasomal degradation. Specific kelch domain-containing F-Box (KFB) proteins as components of ubiquitin E3 ligase directly interact with PAL or CHS, leading to polyubiquitinated PAL and CHS, which in turn influences phenylpropanoid and flavonoid production. Although phenylpropanoids are vital for tomato nutritional value and stress responses, the post-translational regulation of PAL and CHS in tomato remains unknown. We identified 31 putative KFB-encoding genes in the tomato genome. Our homology analysis and phylogenetic study predicted four PAL-interacting SlKFBs, while SlKFB18 was identified as the sole candidate for the CHS-interacting KFB. Consistent with their homolog function, the predicted four PAL-interacting SlKFBs function in PAL degradation. Surprisingly, SlKFB18 did not interact with tomato CHS and the overexpression or knocking out of SlKFB18 did not affect phenylpropanoid contents in tomato transgenic lines, suggesting its irreverence with flavonoid metabolism. Our study successfully discovered the post-translational regulatory machinery of PALs in tomato while highlighting the limitation of relying solely on a homology-based approach to predict interacting partners of F-box proteins.

The Spatial Extracellular Proteomic Tumor Microenvironment Distinguishes Molecular Subtypes of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) mortality rates continue to increase faster than those of other cancer types due to high heterogeneity, which limits diagnosis and treatment. Pathological and molecular subtyping have identified that HCC tumors with poor outcomes are characterized by intratumoral collagenous accumulation. However, the translational and post-translational regulation of tumor collagen, which is critical to the outcome, remains largely unknown. Here, we investigate the spatial extracellular proteome to understand the differences associated with HCC tumors defined by Hoshida transcriptomic subtypes of poor outcome (Subtype 1; S1; n = 12) and better outcome (Subtype 3; S3; n = 24) that show differential stroma-regulated pathways. Collagen-targeted mass spectrometry imaging (MSI) with the same-tissue reference libraries, built from untargeted and targeted LC-MS/MS was used to spatially define the extracellular microenvironment from clinically-characterized, formalin-fixed, paraffin-embedded tissue sections. Collagen α-1(I) chain domains for discoidin-domain receptor and integrin binding showed distinctive spatial distribution within the tumor microenvironment. Hydroxylated proline (HYP)-containing peptides from the triple helical regions of fibrillar collagens distinguished S1 from S3 tumors. Exploratory machine learning on multiple peptides extracted from the tumor regions could distinguish S1 and S3 tumors (with an area under the receiver operating curve of ≥0.98; 95% confidence intervals between 0.976 and 1.00; and accuracies above 94%). An overall finding was that the extracellular microenvironment has a high potential to predict clinically relevant outcomes in HCC.

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

Insights into the role of FoxL2 in tebuconazole-induced male- biased sex differentiation of zebrafish

Tebuconazole (TEB) is a commonly used fungicide that inhibits the aromatase Cyp19A and downregulates the transcription factor forkhead box L2 (FoxL2), leading to male-biased sex differentiation in zebrafish larvae. However, the specific mechanism by which FoxL2 functions following TEB exposure remains unclear. In this study, the phosphorylation sites and kinase-specific residues in zebrafish FoxL2 protein (zFoxL2) were predicted. Subsequently, recombinant zFoxL2 was prepared via prokaryotic expression, and a polyclonal rabbit-anti-zFoxL2 antibody was generated. Zebrafish fibroblast (ZF4) cells were exposed to 100-μM TEB alone for 8 h, after which changes in the expression of genes involved in the foxl2 regulatory pathway (akt1, pi3k, cyp19a1b, c/ebpb and sox9a) were detected. When co-exposed to 1-μM estradiol and 100-μM TEB, the expression of these key genes tended to be restored. Interestingly, TEB did not affect the expression of the foxl2 gene or protein but it significantly suppressed the phosphorylation of FoxL2 (pFoxL2) at serine 238 (decreased by 43.64 %, p = 0.009). Co-immunoprecipitation assays showed that, following exposure to 100-μM TEB, the total precipitated proteins in ZF4 cells decreased by 17.02 % (p = 0.029) and 31.39 % (p = 0.027) in the anti-zFoxL2 antibody group and anti-pFoxL2 (ser238) antibody group, respectively, indicating that TEB suppressed the capacity of the FoxL2 protein to bind to other proteins via repression of its own phosphorylation. The pull-down assay confirmed this conclusion. This study preliminarily elucidated that the foxl2 gene functions via post-translational regulation through hypophosphorylation of its encoded protein during TEB-induced male-biased sex differentiation.

Insights into the role of FoxL2 in tebuconazole-induced male- biased sex differentiation of zebrafish

Tebuconazole (TEB) is a commonly used fungicide that inhibits the aromatase Cyp19A and downregulates the transcription factor forkhead box L2 (FoxL2), leading to male-biased sex differentiation in zebrafish larvae. However, the specific mechanism by which FoxL2 functions following TEB exposure remains unclear. In this study, the phosphorylation sites and kinase-specific residues in zebrafish FoxL2 protein (zFoxL2) were predicted. Subsequently, recombinant zFoxL2 was prepared via prokaryotic expression, and a polyclonal rabbit-anti-zFoxL2 antibody was generated. Zebrafish fibroblast (ZF4) cells were exposed to 100-μM TEB alone for 8 h, after which changes in the expression of genes involved in the foxl2 regulatory pathway (akt1, pi3k, cyp19a1b, c/ebpb and sox9a) were detected. When co-exposed to 1-μM estradiol and 100-μM TEB, the expression of these key genes tended to be restored. Interestingly, TEB did not affect the expression of the foxl2 gene or protein but it significantly suppressed the phosphorylation of FoxL2 (pFoxL2) at serine 238 (decreased by 43.64 %, p = 0.009). Co-immunoprecipitation assays showed that, following exposure to 100-μM TEB, the total precipitated proteins in ZF4 cells decreased by 17.02 % (p = 0.029) and 31.39 % (p = 0.027) in the anti-zFoxL2 antibody group and anti-pFoxL2 (ser238) antibody group, respectively, indicating that TEB suppressed the capacity of the FoxL2 protein to bind to other proteins via repression of its own phosphorylation. The pull-down assay confirmed this conclusion. This study preliminarily elucidated that the foxl2 gene functions via post-translational regulation through hypophosphorylation of its encoded protein during TEB-induced male-biased sex differentiation.

Participation of CWINV and SUS Genes in Sucrose Utilization in the Disruption of Cambium Derivatives Differentiation of Silver Birch.

The mechanisms that control the accumulation of woody biomass are of great interest to the study. Invertase and sucrose synthase are enzymes that are vital for distributing carbon in various biosynthetic pathways. Karelian birch (Betula pendula var. carelica) is a form of silver birch (B. pendula Roth) and is characterized by disruption of the differentiation of cambium derivatives towards both the xylem and phloem, which leads to a change in the proportion of the conducting tissues' structural elements and the figured wood formation. We researched the expression profiles of genes encoding sucrose-cleaving enzymes (CWINV and SUS gene families) and genes encoding CVIF protein, which is responsible for the post-translational regulation of the cell wall invertase activity. In our study, 16-year-old common silver birch (Betula pendula var. pendula) and Karelian birch were used for sampling non-figured and figured trunk section tissues, respectively. Samples were selected for the research based on the radial vector: non-conductive, conductive phloem, cambial zone - differentiating xylem - mature xylem. The enzyme's activity was investigated by biochemical methods. RT-PCR method was used to determine the level of gene expression. Anatomical and morphological methods were used to determine the stage of differentiation of xylem cambial derivatives. Our research revealed a shift in the composition of xylem components in figured Karelian birch, characterized by increased parenchymatization and reduced vessel quantity. In all studied trunk tissues of Karelian birch, compared with common silver birch, an increase in the expression of the CWINV gene family and the SUS3 gene and a decrease in the expression of SUS4 were shown. Therefore, the increase in parenchymatization in figured Karelian birch is linked to a shift in sucrose metabolism towards the apoplastic pathway, indicated by a higher cell wall invertase activity and gene expression. The expression of the SUS4 gene correlates with the decrease in xylem increments and vessel proportion. The research findings will enhance our understanding of how sucrose breaking enzymes regulate secondary growth in woody plants and aid in developing practical timber cultivation methods.

Exploring the role of microRNAs as diagnostic and prognostic biomarkers in canine mammary tumors.

Canine mammary tumors (CMTs) represent a significant health concern in dogs, with a high incidence among intact female dogs. CMTs are a promising comparative model for human breast cancer, due to sharing several pathophysiological features. Additionally, CMTs have a strong genetic correlation with their human counterpart, including the expression of microRNAs (miRNAs). MiRNAs are a class of non-coding RNAs that play important roles in post-translational regulation of gene expression, being implicated in carcinogenesis, tumor progression, and metastasis. Moreover, miRNAs hold promise as diagnostic, prognostic, and metastatic biomarkers. Understanding the molecular mechanisms underlying CMTs is crucial for improving diagnosis, prognosis, and monitoring of treatments. Herein, we provide a comprehensive overview of the current knowledge on miRNAs in CMTs, highlighting their roles in carcinogenesis and their potential as biomarkers. Additionally, we highlight the current limitations and critically discuss the overarching challenges in this field, emphasizing the need for future research to translate miRNA findings into veterinary clinical practice.

Rab7 GTPase-Mediated Stress Signaling Enhances Salinity Tolerance in AlRabring7 Tobacco Transgenics by Modulating Physio-Biochemical parameters

The RING-type E3 ligases play a significant role in stress signaling, primarily through post-translational regulation. Ubiquitination is a crucial post-translational modification that regulates the turnover and activity of proteins. The overexpression of AlRabring7, RING-HC E3 Ub ligase in tobacco provides insights into the regulation of salinity and ABA signaling in transgenic tobacco. The seed germination potential of AlRabring7 transgenics was higher than WT, with NaCl and ABA treatments. The transgenics showed improved morpho-physio-biochemical parameters in response to salinity and ABA treatments. The photosynthetic pigments, soluble sugars, reducing sugars and proline increased in transgenics in response to NaCl and ABA treatments. The decreased ROS accumulation in transgenics on NaCl and ABA treatments can be co-related to improved activity of enzymatic and non- enzymatic antioxidants. The potential of transgenics to maintain ABA levels with ABA treatment, highlights the active participation of ABA feedback loop mechanism. Interestingly, the ability of AlRabring7 transgenics to upregulate Rab7 protein, suggests its role in facilitating vacuolar transport. Furthermore, the improved potassium accumulation and reduced sodium content indicate an efficient ion regulation mechanism in transgenic plants facilitating higher stomatal opening. The expression of downstream ion transporter (NbNHX1 and NbVHA1), ABA signaling (NbABI2 and NbABI5) and vesicle trafficking (NbMON1) responsive genes were upregulated with stress. The present study, reports that AlRabring7 participates in maintaining vacuolar transport, ion balance, ROS homeostasis, stomatal regulation through activation of Rab7 protein and regulation of downstream stress-responsive during stress. This emphasizes the potential of AlRabring7 gene for improved performance and resilience in challenging environments.

LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

Disentangling the splicing factor programs underlying complex molecular phenotypes.

The regulation of exon inclusion through alternative splicing tunes the cell's behavior by increasing the functional diversity of the transcriptome and the proteome. Splicing factors work in concert to generate gene isoform pools that contribute to cell phenotypes yet their activity is controlled by multiple regulatory and signaling layers. This hinders identification of functional, phenotype-specific splicing factors using traditional single-omic measurements, such as their mutational state or expression. To address this challenge, we propose repurposing the virtual inference of protein activity by enriched regulon analysis (VIPER) to measure splicing factor activity solely from their downstream exon transcriptomic inclusion signatures. This approach is effective in assessing the effect of co-occurring splicing factor perturbations, as well as their post-translational regulation. As proof of concept, we dissect recurrent splicing factor programs underlying tumorigenesis including aberrantly activated factors acting as oncogenes and inactivated ones acting as tumor suppressors, which are undetectable by more conventional methodologies. Activation and inactivation of these cancer splicing programs effectively stratifies overall survival, as well as cancer hallmarks such as proliferation and immune evasion. Altogether, repurposing network-based inference of protein activity for splicing factor networks distills common, functionally relevant splicing programs in otherwise heterogeneous molecular contexts.

Omeprazole Induces CYP3A4 mRNA Expression but Not CYP3A4 Protein Expression in HepaRG Cells.

Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug-drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.

Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.

Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes and distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) that dramatically improves the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely-matched control promoters, integrated together into the genome at single copy. To test our ability to capture post-transcriptional and post-translational regulation through sequencing, we screened for genes that affected nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq screens accurately capture the known components of well-studied RNA and protein quality control pathways with minimal background. These results demonstrate the precision and versatility of CiBER-seq for dissecting the genetic networks controlling cellular behaviors.

The new kids on the block: RNA-binding proteins regulate autophagy in disease.

Mammalian autophagy is a highly regulated and conserved cellular homeostatic process. Its existence allows the degradation of self-components to mediate cell survival in different stress conditions. Autophagy is involved in the regulation of cellular metabolic needs, protecting the cell or tissue from starvation through the degradation and recycling of cytoplasmic materials and organelles to basic molecular building blocks. It also plays a critical role in eliminating damaged or harmful proteins, organelles, and intracellular pathogens. Thus, a deterioration of the process may result in pathological conditions, such as aging-associated disorders and cancer. Understanding the crucial role of autophagy in maintaining the normal physiological function of cells, tissue, or organs has led to copious and expansive research regarding the regulation of this process. So far, most of the research has revolved around transcriptional and post-translational regulation. Here, we discuss the regulation of autophagy-related (ATG) mRNA transcripts by RNA-binding proteins (RBPs). This analysis focuses on how RBPs modulate autophagy in disease. A deeper understanding of the involvement of RBPs in autophagy can facilitate further research and treatment of a variety of human diseases.

Mitochondrial Chaperone Code: Just warming up.

More than 99% of the mitochondrial proteome is encoded by the nucleus and requires refolding following import. Therefore, mitochondrial proteins require the coordinated action of molecular chaperones for their folding and activation. Several heat shock protein (Hsp) molecular chaperones, including members of the Hsp27, Hsp40/70, and Hsp90 families, as well as the chaperonin complex Hsp60/10 have an established role in mitochondrial protein import and folding. The "Chaperone Code" describes the regulation of chaperone activity by dynamic post-translational modifications; however, little is known about the post-translational regulation of mitochondrial chaperones. Dissecting the regulation of chaperone function is essential for understanding their differential regulation in pathogenic conditions and the potential development of efficacious therapeutic strategies. Here, we summarize the recent literature on post-translational regulation of mitochondrial chaperones, the consequences for mitochondrial function, and potential implications for disease.

Maf1 phosphorylation is regulated through the action of prefoldin-like Bud27 on PP4 phosphatase in Saccharomyces cerevisiae.

Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here, we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like protein on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53.

Protective effect of melatonin against methamphetamine-induced attention deficits through miR-181/SIRT1 axis in the prefrontal cortex.

Methamphetamine (METH) is an addictive psychostimulant with deleterious effects on the central nervous system. Chronic use of METH in high doses impairs cognition, attention and executive functions, but the underlying mechanisms are still unclear. Sirtuin 1 (SIRT1) is a post-translational regulator that is downregulated following METH neurotoxicity. Melatonin is a neuroprotective hormone that enhances mitochondrial metabolism. Here, we evaluated the effect of melatonin on METH-induced attention deficits disorder and the involvement of the miR-181/SIRT1 axis in melatonin neuroprotection. METH at a dose of 5mg/kg was injected for 21 consecutive days. The animals were assigned to receive either melatonin or the vehicle after METH injections. Attention levels were evaluated with abject-based attention test. In the prefrontal cortex, the expression levels of miR-181a-5p, SIRT1, p53 and CCAR2, as well as the mtDNA copy numbers were evaluated using qRT-PCR and western blotting. The outcomes revealed that melatonin treatment following METH injections improved METH-induced attention deficits. METH toxicity can be associated with changes in the miR-181/SIRT1 axis, elevated levels of p53 and COXII, and decreased levels of mtDNA in the prefrontal cortex of adult rats. Interestingly, administration of melatonin can improve the expression of these molecules and reduces the toxic effects of METH. Melatonin ameliorated the neurotoxicity of METH in the prefrontal cortex and the miR-181/SIRT1 axis is involve in the protective effects of melatonin. However, melatonin can be potentially administrated to improve attention impairment in METH use disorders.

A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

Protein Tyrosine Phosphatase Non-receptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 post-translational regulation. Here we characterize a phosphorylation site at Ser325 situated C-terminal to the catalytic domain of PTPN22, and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by Glycogen Synthase Kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9 mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p and Moira (SWIRM) domain binding proteins, and next-generation sequencing (NGS) revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by Deuterium Exchange Mass Spectrometry (DX/MS) and Nuclear Magnetic Resonance (NMR) spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22. Significance statement The tyrosine phosphatase PTPN22 serves as a negative regulator in T cells, and its phosphorylation is a major regulatory process for controlling its function. Here, we uncovered a novel phosphorylation site at Ser325 on PTPN22 that allosterically regulates its activity leading to impaired TCR-dependent pathways. Biophysical methods identify multiple regions affected upon Ser325 phosphorylation, which can be the basis for future mechanistic studies of PTPN22 activators or inhibitors.

MEG3 in hematologic malignancies: from the role of disease biomarker to therapeutic target.

Maternally expressed gene 3 (MEG3) is a noncoding RNA that is known as a tumor suppressor in solid cancers. Recently, a line of studies has emphasized its potential role in hematological malignancies in terms of tumorigenesis, metastasis, and drug resistance. Similar to solid cancers, MEG3 can regulate various cancer hallmarks via sponging miRNA, transcriptional, or posttranslational regulation mechanisms, but may regulate different key elements. In contrast with solid cancers, in some subtypes of leukemia, MEG3 has been found to be upregulated and oncogenic. In this review, we systematically describe the role and underlying mechanisms of MEG3 in multiple types of hematological malignancies. Particularly, we highlight the role of MEG3 in drug resistance and as a novel therapeutic target.

MTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells.

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.

MTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.