RESUMEN
Tks4 is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as cellular motility, reactive oxygen species-dependent processes, and embryonic development. It is also implicated in a rare developmental disorder, Frank-ter Haar syndrome. Loss of Tks4 resulted in the induction of an EMT-like process, with increased motility and overexpression of EMT markers in colorectal carcinoma cells. In this work, we explored the broader effects of deletion of Tks4 on the gene expression pattern of HCT116 colorectal carcinoma cells by transcriptome sequencing of wild-type and Tks4 knockout (KO) cells. We identified several protein coding genes with altered mRNA levels in the Tks4 KO cell line, as well as a set of long non-coding RNAs, and confirmed these changes with quantitative PCR on a selected set of genes. Our results show a significant perturbation of gene expression upon the deletion of Tks4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signaling.
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Neoplasias del Colon , Anomalías Craneofaciales , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transducción de Señal/genética , Neoplasias del Colon/genética , Anomalías Craneofaciales/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transición Epitelial-MesenquimalRESUMEN
Colon cancer is a leading cause of death worldwide. Identification of new molecular factors governing the invasiveness of colon cancer holds promise in developing screening and targeted therapeutic methods. The Tyrosine Kinase Substrate with four SH3 domains (TKS4) and the CD2-associated protein (CD2AP) have previously been linked to dynamic actin assembly related processes and cancer cell migration, although their co-instructive role during tumor formation remained unknown. Therefore, this study was designed to investigate the TKS4-CD2AP interaction and study the interdependent effect of TKS4/CD2AP on oncogenic events. We identified CD2AP as a novel TKS4 interacting partner via co-immunoprecipitation-mass spectrometry methods. The interaction was validated via Western blot (WB), immunocytochemistry (ICC) and proximity ligation assay (PLA). The binding motif of CD2AP was explored via peptide microarray. To uncover the possible cooperative effects of TKS4 and CD2AP in cell movement and in epithelial-mesenchymal transition (EMT), we performed gene silencing and overexpressing experiments. Our results showed that TKS4 and CD2AP form a scaffolding protein complex and that they can regulate migration and EMT-related pathways in HCT116 colon cancer cells. This is the first study demonstrating the TKS4-CD2AP protein-protein interaction in vitro, their co-localization in intact cells, and their potential interdependent effects on partial-EMT in colon cancer.
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Neoplasias del Colon , Transición Epitelial-Mesenquimal , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Neoplasias del Colon/genética , Proteínas del Citoesqueleto/metabolismoRESUMEN
Tyrosine kinase substrate with four SH3 domains (Tks4) scaffold protein plays roles in cell migration and podosome formation and regulates systemic mechanisms such as adult bone homeostasis and adipogenesis. Mutations in the Tks4 gene (SH3PXD2b) cause a rare developmental disorder called Frank-Ter Haar syndrome (FTHS), which leads to heart abnormalities, bone tissue defects, and reduced adiposity. We aimed to produce a human stem cell-based in vitro FTHS model system to study the effects of the loss of the Tks4 protein in different cell lineages and the accompanying effects on the cell signalome. To this end, we used CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas9)) to knock out the SH3PXD2b gene in the HUES9 human embryonic stem cell line (hESC), and we obtained stable homo- and heterozygous knock out clones for use in studying the potential regulatory roles of Tks4 protein in embryonic stem cell biology. Based on pluripotency marker measurements and spontaneous differentiation capacity assays, we concluded that the newly generated Tks4-KO HUES9 cells retained their embryonic stem cell characteristics. We propose that the Tks4-KO HUES9 cells could serve as a tool for further cell differentiation studies to investigate the involvement of Tks4 in the complex disorder FTHS. Moreover, we successfully differentiated all of the clones into mesenchymal stem cells (MSCs). The derived MSC cultures showed mesenchymal morphology and expressed MSC markers, although the expression levels of mesodermal and osteogenic marker genes were reduced, and several EMT (epithelial mesenchymal transition)-related features were altered in the Tks4-KO MSCs. Our results suggest that the loss of Tks4 leads to FTHS by altering cell lineage differentiation and cell maturation processes, rather than by regulating embryonic stem cell potential.
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Cardiopatías Congénitas , Células Madre Embrionarias Humanas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Niño , Anomalías Craneofaciales , Discapacidades del Desarrollo/genética , Cardiopatías Congénitas/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Osteocondrodisplasias/congénito , Enfermedades RarasRESUMEN
Somatic mutations in the RAS genes are frequent in human tumors, especially in pancreatic, colorectal, and non-small-cell lung cancers. Such mutations generally decrease the ability of Ras to hydrolyze GTP, maintaining the protein in a constitutively active GTP-bound form that drives uncontrolled cell proliferation. Efforts to develop drugs that target Ras oncoproteins have been unsuccessful. Recent emerging data suggest that Ras regulation is more complex than the scientific community has believed for decades. In this review, we summarize advances in the "textbook" view of Ras activation. We also discuss a novel type of Ras regulation that involves direct phosphorylation and dephosphorylation of Ras tyrosine residues. The discovery that pharmacological inhibition of the tyrosine phosphoprotein phosphatase SHP2 maintains mutant Ras in an inactive state suggests that SHP2 could be a novel drug target for the treatment of Ras-driven human cancers.
Asunto(s)
Neoplasias/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tirosina/metabolismo , Animales , Genes ras , Humanos , Neoplasias/genética , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)/genética , Tirosina/genéticaRESUMEN
KRAS is one of the most commonly mutated oncogene and a negative predictive factor for a number of targeted therapies. Therefore, the development of targeting strategies against mutant KRAS is urgently needed. One potential strategy involves disruption of K-Ras membrane localization, which is necessary for its proper function. In this review, we summarize the current data about the importance of membrane-anchorage of K-Ras and provide a critical evaluation of this targeting paradigm focusing mainly on prenylation inhibition. Additionally, we performed a RAS mutation-specific analysis of prenylation-related drug sensitivity data from a publicly available database ( https://depmap.org/repurposing/ ) of three classes of prenylation inhibitors: statins, N-bisphosphonates, and farnesyl-transferase inhibitors. We observed significant differences in sensitivity to N-bisphosphonates and farnesyl-transferase inhibitors depending on KRAS mutational status and tissue of origin. These observations emphasize the importance of factors affecting efficacy of prenylation inhibition, like distinct features of different KRAS mutations, tissue-specific mutational patterns, K-Ras turnover, and changes in regulation of prenylation process. Finally, we enlist the factors that might be responsible for the large discrepancy between the outcomes in preclinical and clinical studies including methodological pitfalls, the incomplete understanding of K-Ras protein turnover, and the variation of KRAS dependency in KRAS mutant tumors.
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Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Antineoplásicos/farmacología , Genes ras , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/genética , Prenilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
The genetic alterations in cancer cells are tightly linked to signaling pathway dysregulation. Ras is a key molecule that controls several tumorigenesis-related processes, and mutations in RAS genes often lead to unbiased intensification of signaling networks that fuel cancer progression. In this article, we review recent studies that describe mutant Ras-regulated signaling routes and their cross-talk. In addition to the two main Ras-driven signaling pathways, i.e., the RAF/MEK/ERK and PI3K/AKT/mTOR pathways, we have also collected emerging data showing the importance of Ras in other signaling pathways, including the RAC/PAK, RalGDS/Ral, and PKC/PLC signaling pathways. Moreover, microRNA-regulated Ras-associated signaling pathways are also discussed to highlight the importance of Ras regulation in cancer. Finally, emerging data show that the signal alterations in specific cell types, such as cancer stem cells, could promote cancer development. Therefore, we also cover the up-to-date findings related to Ras-regulated signal transduction in cancer stem cells.
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Mutación , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Humanos , Transducción de Señal/genéticaRESUMEN
Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana's Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas Intrínsecamente Desordenadas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Estrés Fisiológico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Intrínsecamente Desordenadas/genética , Chaperonas Moleculares/genética , Presión Osmótica , Proteínas de Plantas/genéticaRESUMEN
The scaffold protein Tks4 is a member of the p47phox-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called "tandem SH3") and a proline-rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.
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Proteínas Adaptadoras Transductoras de Señales , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Humanos , Modelos Moleculares , Dominios Proteicos Ricos en Prolina , Unión Proteica , Dominios Homologos srcRESUMEN
Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr89 and Tyr134 in ABL1 or the homologous residues Tyr116 and Tyr161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome.
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Tirosina/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Humanos , Ligandos , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-abl/metabolismo , Dispersión del Ángulo PequeñoRESUMEN
Scaffold proteins are typically thought of as multi-domain "bridging molecules." They serve as crucial regulators of key signaling events by simultaneously binding multiple participants involved in specific signaling pathways. In the case of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) binding, the activated EGFR contacts cytosolic SRC tyrosine-kinase, which then becomes activated. This process leads to the phosphorylation of SRC-substrates, including the tyrosine kinase substrates (TKS) scaffold proteins. The TKS proteins serve as a platform for the recruitment of key players in EGFR signal transduction, promoting cell spreading and migration. The TKS4 and the TKS5 scaffold proteins are tyrosine kinase substrates with four or five SH3 domains, respectively. Their structural features allow them to recruit and bind a variety of signaling proteins and to anchor them to the cytoplasmic surface of the cell membrane. Until recently, TKS4 and TKS5 had been recognized for their involvement in cellular motility, reactive oxygen species-dependent processes, and embryonic development, among others. However, a number of novel functions have been discovered for these molecules in recent years. In this review, we attempt to cover the diverse nature of the TKS molecules by discussing their structure, regulation by SRC kinase, relevant signaling pathways, and interaction partners, as well as their involvement in cellular processes, including migration, invasion, differentiation, and adipose tissue and bone homeostasis. We also describe related pathologies and the established mouse models.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Diferenciación Celular , Movimiento Celular , Homeostasis , Podosomas/fisiología , Humanos , Transducción de SeñalRESUMEN
The nonreceptor tyrosine kinase Src is a central component of the epidermal growth factor (EGF) signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues 466-474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues 508-511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to a synergistic enhancement of binding strength and a highly stable, "bidentate"-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least 3 h in living cells. We conclude that the interaction of Tks4 with Src may result in the long term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Dominios Homologos src , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Humanos , Familia-src Quinasas/químicaRESUMEN
Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular processes and are extensively involved in the development of different cancers; including leukemias. As one of the accepted methods of lncRNA function is affecting chromatin structure; lncRNA binding has been shown for different chromatin modifiers. Histone lysine methyltransferases (HKMTs) are also subject of lncRNA regulation as demonstrated for example in the case of Polycomb Repressive Complex 2 (PRC2). Mixed Lineage Leukemia (MLL) proteins that catalyze the methylation of H3K4 have been implicated in several different cancers; yet many details of their regulation and targeting remain elusive. In this work we explored the RNA binding capability of two; so far uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein. We demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not; are capable of binding to different RNA constructs in vitro. To our knowledge, these findings are the first to indicate that an MLL protein itself is capable of lncRNA binding.
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Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , Proteínas de Unión al ADN/genética , Proteínas Intrínsecamente Desordenadas/genética , Modelos Biológicos , Unión Proteica , Estructura Secundaria de Proteína , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin-GnRH-III conjugate (GnRH-III-[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin-GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates.
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Mesenchymal stem or stromal cells (MSCs) act on different components of the immune response including macrophages (MΦs). Therefore this study has been committed to explore how MSCs may modify the effect of MΦ polarization upon an inductive environment using mouse bone marrow (BM)-derived "naïve", unpolarized MΦs. Phagocytosis of various MΦ subtypes was different since M1 and M2b showed poorer, while M2a higher rate of phagocytosis. MSCs significantly promoted yeast ingestion by M1 and M2b and diminished it by M2a cells. Under polarizing conditions, MSCs profoundly affected the TNFα production of MΦ subtypes since M1 and M2b MΦs produced less and M2a produced higher amount of TNFα while the amount of IL-10 was not affected. The most striking effect of MSCs was registered on M2b cells since the inflammatory TNFα dominance remarkably shifted to the immunosuppressive IL-10. Prepolarized M1 cells readily converted to M2a and M2b states when polarizing conditions changed from M1 to M2a or M2b induction, respectively. Repolarizing from M1 to M2a resulted in the decline of IL-10 and TNFα and defined elevation of Ym1 similar to levels characteristic to M2a primarily polarized from naïve BM-MΦs. Similarly, polarization of M1 to M2b MΦs was successful showing increase in IL-10 and reduction in TNFα levels characteristic to M2b cells. However, when co-culturing with MSCs, M1-M2a or M1-M2b transition was not affected. Crosstalk between MΦs and MSCs depended on PGE-2 since COX-2 inhibition reduced the effect of MSCs to establish an IL-10-dominant cytokine production by MΦs.
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Polaridad Celular , Macrófagos/citología , Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/citología , Separación Celular , Citocinas/biosíntesis , Dinoprostona/metabolismo , Interleucina-10/biosíntesis , Macrófagos/metabolismo , Macrófagos Peritoneales/citología , Ratones Endogámicos C57BL , Fagocitosis , Saccharomyces cerevisiae/citología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND: The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. RESULTS: To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr(280) (pTyr(280)) and pTyr(305). These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr(280) and pTyr(305) on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. CONCLUSIONS: TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Oncogénicas/metabolismo , Fosfoproteínas/metabolismo , Mapas de Interacción de Proteínas , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Secuencia de Aminoácidos , Animales , Células Cultivadas , Células HEK293 , Humanos , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/análisis , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Oncogénicas/análisis , Fosfoproteínas/análisis , Dominios Homologos srcRESUMEN
BACKGROUND: Cells deploy quality control mechanisms to remove damaged or misfolded proteins. Recently, we have reported that a mutation (R43W) in the Frank-ter Haar syndrome protein Tks4 resulted in aberrant intracellular localization. RESULTS: Here we demonstrate that the accumulation of Tks4(R43W) depends on the intact microtubule network. Detergent-insoluble Tks4 mutant colocalizes with the centrosome and its aggregate is encaged by the intermediate filament protein vimentin. Both the microtubule inhibitor nocodazole and the histone deacetylase inhibitor Trichostatin A inhibit markedly the aggresome formation in cells expressing Tks4(R43W). Finally, pretreatment of cells with the proteasome inhibitor MG132 markedly increases the level of aggresomes formed by Tks4(R43W). Furthermore, two additional mutant Tks4 proteins (Tks4(1-48) or Tks4(1-341)) have been investigated. Whereas the shorter Tks4 mutant, Tks4(1-48), shows no expression at all, the longer Tks4 truncation mutant accumulates in the nuclei of the cells. CONCLUSIONS: Our results suggest that misfolded Frank-ter Haar syndrome protein Tks4(R43W) is transported via the microtubule system to the aggresomes. Lack of expression of Tks4(1-48) or aberrant intracellular expressions of Tks4(R43W) and Tks4(1-341) strongly suggest that these mutations result in dysfunctional proteins which are not capable of operating properly, leading to the development of FTHS.
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Proteínas Adaptadoras Transductoras de Señales/genética , Anomalías Craneofaciales/genética , Cardiopatías Congénitas/genética , Microtúbulos/patología , Osteocondrodisplasias/congénito , Mutación Puntual , Agregación Patológica de Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Chlorocebus aethiops , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Pliegue de Proteína , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
The epithelial-to-mesenchymal transition (EMT) represents a hallmark event in the evolution of lung cancer. This work aims to study a recently described EMT-regulating protein, Tks4, and to explore its potential as a prognostic biomarker in non-small cell lung cancer. In this study, we used CRISPR/Cas9 method to knockout (KO) Tks4 to study its functional roles in invadopodia formation, migration, and regulation of EMT marker expressions and we identified Tks4-interacting proteins. Tks4-KO A549 cells exhibited an EMT-like phenotype characterized by elongated morphology and increased expression of EMT markers. Furthermore, analyses of a large-scale lung cancer database and a patient-derived tissue array data revealed that the Tks4 mRNA level was decreased in more aggressive lung cancer stages. To understand the regulatory role of Tks4 in lung cancer, we performed a Tks4-interactome analysis via Tks4 immunoprecipitation-mass spectrometry on five different cell lines and identified CAPZA1 as a novel Tks4 partner protein. Thus, we propose that the absence of Tks4 leads to disruption of a connectome of multiple proteins and that the resulting undocking and likely mislocalization of signaling molecules impairs actin cytoskeleton rearrangement and activates EMT-like cell fate switches, both of which likely influence disease severity.
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Carcinoma de Pulmón de Células no Pequeñas , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares , Humanos , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Movimiento Celular , Plasticidad de la Célula , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Podosomas/metabolismoRESUMEN
Background: Colorectal carcinoma (CRC) has emerged as one of the most widespread cancers and was the third leading cause of cancer-related mortality in 2020. The role of the podosomal protein Tks4 in tumor formation and progression is well established, including its involvement in gastric carcinoma and hepatocellular carcinoma; however, exploration of Tks4 and its associated EMT-regulating interactome in the context of colon cancer remains largely unexplored. Methods: We conducted a comprehensive bioinformatic analysis to investigate the mRNA and protein expression levels of Tks4 and its associated partner molecules (CD2AP, GRB2, WASL, SRC, CTTN, and CAPZA1) across different tumor types. We quantified the expression levels of Tks4 and its partner molecules using qPCR, utilizing a TissueScan colon cancer array. We then validated the usefulness of Tks4 and its associated molecules as biomarkers via careful statistical analyses, including Pearson's correlation analysis, principal component analysis (PCA), multiple logistic regression, confusion matrix analysis, and ROC analysis. Results: Our findings indicate that the co-expression patterns of the seven examined biomarker candidates better differentiate between tumor and normal samples compared with the expression levels of the individual genes. Moreover, variable importance analysis of these seven genes revealed four core genes that yield consistent results similar to the seven genes. Thus, these four core genes from the Tks4 interactome hold promise as potential combined biomarkers for colon adenocarcinoma diagnosis and prognosis. Conclusion: Our proposed biomarker set from the Tks4 interactome shows promising sensitivity and specificity, aiding in colon cancer prevention and diagnosis.
RESUMEN
Covalent drugs might bear electrophiles to chemically modify their targets and have the potential to target previously undruggable proteins with high potency. Covalent binding of drug-size molecules includes a noncovalent recognition provided by secondary interactions and a chemical reaction leading to covalent complex formation. Optimization of their covalent mechanism of action should involve both types of interactions. Noncovalent and covalent binding steps can be characterized by an equilibrium dissociation constant (KI) and a reaction rate constant (kinact), respectively, and they are affected by both the warhead and the scaffold of the ligand. The relative contribution of these two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620 that was equipped with four different warheads and a previously described KRAS-specific basic side chain. Combining these structural changes, we have synthesized novel covalent KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by various biophysical and biochemical assays. These data allowed us to dissect the effect of scaffold and warhead on the noncovalent and covalent binding event. Our results revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C inhibition, the basic side chain has little effect on either binding step, and warheads affect the covalent reactivity but not the noncovalent binding. This type of analysis helps identify structural determinants of efficient covalent inhibition and may find use in the design of covalent agents.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/química , Humanos , Unión Proteica , Mutación , LigandosRESUMEN
Fragment screening is a popular strategy of generating viable chemical starting points especially for challenging targets. Although fragments provide a better coverage of chemical space and they have typically higher chance of binding, their weak affinity necessitates highly sensitive biophysical assays. Here, we introduce a screening concept that combines evolutionary optimized fragment pharmacophores with the use of a photoaffinity handle that enables high hit rates by LC-MS-based detection. The sensitivity of our screening protocol was further improved by a target-conjugated photocatalyst. We have designed, synthesized, and screened 100 diazirine-tagged fragments against three benchmark and three therapeutically relevant protein targets of different tractability. Our therapeutic targets included a conventional enzyme, the first bromodomain of BRD4, a protein-protein interaction represented by the oncogenic KRasG12D protein, and the yet unliganded N-terminal domain of the STAT5B transcription factor. We have discovered several fragment hits against all three targets and identified their binding sites via enzymatic digestion, structural studies and modeling. Our results revealed that this protocol outperforms screening traditional fully functionalized and photoaffinity fragments in better exploration of the available binding sites and higher hit rates observed for even difficult targets.