Grb2 Research Articles

Circ_0026134 regulates the miR-1270/GRB2 pathway to affect the radiosensitivity of hepatoma cells

Objective: To investigate whether circular RNA 0026134 (circ_0026134) affects the radiosensitivity of hepatoma cells by regulating the miR-1270/growth factor receptor-bound protein 2 (GRB2) pathway. Methods: Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of circ_0026134, miR-1270, and GRB2 in liver cancer tissues and cells. Bioinformatics analysis, a dual-luciferase gene reporter assay, RT-qPCR, and western blot were used to analyze the targeting relationships between circ_0026134 and miR-1270 and miR-1270 and GRB2. The effects of circ_0026134, miR-1270, and GRB2 expression combined with 6 Gy on the proliferation, invasion, migration, and apoptosis of Huh7 and SK-HEP-1 cells were detected by a cell counting kit, a transwell assay, a scratch assay, and flow cytometry. The tumorigenesis experiment was used to detect the effect of silencing circ_0026134 in nude mice. Measurement data are expressed as the mean ± standard deviation. The independent sample t-test was used for comparison between two groups, and the one-way analysis of variance and SNK-q test were used for comparison between multiple groups. P < 0.05 was considered statistically significant. Results: The expression levels of circ_0026134 and GRB2, Huh7, and SK-HEP-1 cells in liver cancer tissues were significantly increased, while the expression levels of miR-1270 were significantly decreased (P < 0.05). The expression of circ_0026134 in Huh7 and SK-HEP-1 decreased significantly after radiotherapy (P < 0.05). circ_0026134 binds directly to miR-1270 and negatively regulates miR-1270 expression (P < 0.05). miR-1270 binds directly to GRB2 and negatively regulates GRB2 expression (P < 0.05). 6 Gy radiation significantly inhibited the proliferation, migration, and invasion of Huh7 and SK-HEP-1 cells and induced apoptosis (P < 0.05). Silencing circ_0026134 or overexpression of miR-1270 significantly enhanced the anti-proliferation, anti-migration, invasion, and pro-apoptosis effects of 6 Gy treatment on hepatoma cells (P < 0.05). Inhibition of miR-1270 significantly weakened the effects of silencing circ_0026134 combined with 6 Gy radiation on proliferation, migration, invasion, and apoptosis of hepatoma cells (P < 0.05). Overexpression of GRB2 significantly weakened the effects of miR-1270 overexpression combined with 6 Gy radiation on proliferation, migration, invasion, and apoptosis of hepatoma cells (P < 0.05). circ_0026134 knockdown significantly delayed tumor growth in vivo (P < 0.05). Conclusion: Silencing circ_0026134 strengthens radiation treatment's anti-proliferation, anti-migration, invasion, and pro-apoptotic effects in hepatoma cells by negatively regulating the miR-1270/GRB2 pathway, thereby enhancing radiosensitivity.

Golgi Protein 73 Promotes Angiogenesis in Hepatocellular Carcinoma.

Golgi protein 73 (GP73), a resident protein of the Golgi apparatus, is notably elevated in hepatocellular carcinoma (HCC). While its critical role in remodeling the tumor microenvironment (TME) is recognized, the intricate mechanisms are not fully understood. This study reveals that GP73 in HCC cells interacts with prolyl hydroxylase-2 (PHD-2) in a competitive manner, thereby impeding the hydroxylation of hypoxia-induced factor-1α (HIF-1α). The effect above promotes the production and secretion of vascular endothelial growth factor A (VEGFA). Moreover, exosomal GP73 derived from HCC cells can be internalized by human umbilical vein endothelial cells (HUVECs) and competitively interact with HECTD1, an E3 ubiquitin ligase targeting growth factor receptor-bound protein 2 (GRB2). This interaction stabilizes GRB2, thereby activating the Ras-mitogen-activated protein kinase (MAPK) signaling pathway. Consequently, escalated levels of GP73 intensify VEGF production in HCC cells and potentiate mitogenic signaling in vascular endothelial cells, fostering angiogenesis in the TME. Our findings propose that GP73 might serve as a novel target for anti-angiogenic therapy in HCC.

Highly oxidized and rearranged schinortriterpenoids with neuroprotective activity from the stems and leaves of Schisandra chinensis

The stems and leaves (SCSL) are the main byproducts of the Schisandra chinensis cultivation process, and they are worthy of research and utilization from the perspective of medicine and economic benefits. Eighteen (1–18) undescribed highly oxygenated and rearranged schinortriterpenoids (SNTs) and six analogues (19–24), including 14(13 → 12):16(17 → 13)-diabeoschiartane (1–7), 18(13 → 14)-abeoschiartanes (8–12, 19), 18-norschiartane (13), schiartanes (14, 20), 16,17-secopreschisanartanes (15–17, 21–24), lancifoartane (18) skeletons, were isolated from the SCSL. Compounds 1–7 feature a rare 7/5/5-fused carbocyclic core. The structures were established by MS, NMR, single-crystal X-ray diffraction, ECD, and biogenetic considerations. In addition, neuroprotective assays were performed to gain a preliminary understanding of their biological activity. Compounds 2, 6, and 7 showed strong neurite outgrowth-promoting activity with 13.1 %, 12.0 %, and 12.2 % cell differentiation rate (positive group: 15.4 %), respectively. Compounds 1, 2, and 11 at a concentration of 25 μM also had neuroprotective effect on corticosterone (CORT)-induced PC12 cell injury, the cell viability was increased by 21.1 %, 19.5 %, and 24.4 % (positive group: 30.6 %), respectively. Molecular docking analysis and protein–protein interaction (PPI) network revealed that compounds 2, 6, and 7 can exert neuroprotective effects through the regulation of growth factor receptor-bound protein 2 (GRB2). The discovery was beneficial to the high-value utilization of SCSL and the development of natural neuroprotective drugs.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

A Multi-Modal Analysis of Acquired Resistance to Acalabrutinib and Pirtobrutinib Provides Potential Strategies to Augment Treatment Outcome with BTKi Drugs

Introduction: Acalabrutinib (acala), a covalent BTK inhibitor (BTKi), exhibits excellent efficacy and safety in CLL and other B cell malignancies. However, long term efficacy is often compromised by the acquisition of mutations at the acala-binding residue C481, the most prevalent being C481S. Pirtobrutinib (pirto), a reversible BTKi, is active clinically in patients who have progressed on a covalent BTKi with or without C481S mutations. Besides BTK mutations, several additional pathways are hypothesized to contribute to BTKi resistance and may be highly relevant to the sequencing and combining of CLL-directed therapies. To understand acquired resistance to both acala and pirto, we employed multiple approaches, including a genome-wide CRISPR knockout (KO) screen and the generation of BTK mutant drug resistant cell lines. Methods: Genome-wide CRISPR KO screen was performed in the BTKi-sensitive CD79B-MYD88 double mutant ABC-DLBCL cell line TMD8 with 3 guides/gene (best Vienna BioScore). Pooled library-transduced cells at 750x target coverage were cultured for 14 days with sublethal acala or pirto concentrations to achieve ~60% growth inhibition over DMSO. NGS/BAGAL/MAGeCK analyses identified enriched/depleted guides and corresponding resistance/sensitizing genes (FDR&amp;lt;0.1). Hits were validated by single gene KO (Cas9 RNP + 3-plex gRNA nucleofection) and 10-day growth assays with BTKi treatment. BTK mutant cell lines resistant to acala or pirto were derived in TMD8 and the BTKi-sensitive Karpas1718 MZL cell line by culturing through dose escalation. Exome/RNAseq was used to identify BTK mutations. TMD8 BTK C481R cell line was generated by CRISPR-HDR. BTK kinase and pathway activities were assayed by Western/Flow. Results: CRISPR screening results correlated highly between acala and pirto (Pearson's r=0.66), indicating shared pathways that impact response to both BTKi's. Collectively we identified 133 resistance and 57 sensitizing genes that span both known pathways (BCR/NFκB signaling) and novel modulators (autophagy, g-secretase, PBAF complex) of BTKi response. Systematic hit validation by single gene KO confirmed resistance phenotype for 6 genes: TNFAIP3 and TRAF3 negative regulators of canonical/non-canonical NFκB pathways; GRB2 adaptor protein for BCR signaling; PSEN1 in γ-secretase, PHF10 in PBAF complex; and SPOP substrate recognition protein for ubiquitin ligase. Interrogating the Project GENIE patient database identified loss-of-function mutations in these genes (8.6% CLL, 19.3% MCL, 25% MZL), supporting the hypothesis that they may be biomarkers for resistance to both BTKis. Sensitizing genes included CBL adaptor protein, PTPRCAP a CD45 tyrosine phosphatase, and RRAS2 small GTPase; KO of these genes did not alter TMD8 cell growth but enhanced the potency of both BTKis. Genomic analysis of BTKi-adaptive resistant cell lines identified causal BTK mutations, including nonclassical binding site mutation C481F for acala resistance and 2 novel mutations F442L and L460S for pirto resistance. C481F and C481R are kinase-inactive but cells still retain BCR signaling, pointing to BTK scaffolding activity. HCK is a proposed mediator of BTK scaffolding activity (Dhami et al., 2022; Yuan et al., 2022). However, while HCK activation was detected in C481R cells, HCK KO did not impair cell growth. In addition, no HCK activation was detected in C481F cells. Thus, scaffolding activity by C481F/R mutants does not depend on HCK. For the 2 pirto-resistant BTK mutations, F442L is kinase active but has reduced response to pirto, whereas L460S is kinase impaired. All our BTK mutant cell lines were still sensitive to the other BTKi, supporting a rationale for treatment sequencing between BTKi drugs to overcome resistance due to BTK mutations. Conclusions: We uncovered known and novel BTK resistance mutations and demonstrated BTK scaffolding activity independent of HCK, highlighting the need for other strategies to disrupt scaffolding-mediated BCR signaling. Beyond BTK mutations, our CRISPR KO screens illustrate a map of genetic modifiers of BTKi response and point to several potential resistance biomarkers for both acala and pirto. A better understanding of resistance mechanisms in the presence and absence of BTK mutations will help augment the use of BTKi treatments in patients.

Erratum: Growth Factor Receptor Bound Protein 2–Associated Binder 2, a Scaffolding Adaptor Protein, Negatively Regulates Host Immunity against Tuberculosis

Erratum: Growth Factor Receptor Bound Protein 2–Associated Binder 2, a Scaffolding Adaptor Protein, Negatively Regulates Host Immunity against Tuberculosis

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding.

The folding process of multidomain proteins is a highly intricate phenomenon involving the assembly of distinct domains into a functional three-dimensional structure. During this process, each domain may fold independently while interacting with others. The folding of multidomain proteins can be influenced by various factors, including their composition, the structure of each domain, or the presence of disordered regions, as well as the surrounding environment. Misfolding of multidomain proteins can lead to the formation of nonfunctional structures associated with a range of diseases, including cancers or neurodegenerative disorders. Understanding this process is an important step for many biophysical analyses such as stability, interaction, malfunctioning, and rational drug design. One such multidomain protein is growth factor receptor-bound protein 2 (GRB2), an adaptor protein that is essential in regulating cell survival. GRB2 consists of one central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. The SH2 domain interacts with phosphotyrosine regions in other proteins, while the SH3 domains recognize proline-rich regions on protein partners during cell signaling. Here, we combined computational and experimental techniques to investigate the folding process of GRB2. Through computational simulations, we sampled the conformational space and mapped the mechanisms involved by the free energy profiles, which may indicate possible intermediate states. From the molecular dynamics trajectories, we used the energy landscape visualization method (ELViM), which allowed us to visualize a three-dimensional (3D) representation of the overall energy surface. We identified two possible parallel folding routes that cannot be seen in a one-dimensional analysis, with one occurring more frequently during folding. Supporting these results, we used differential scanning calorimetry (DSC) and fluorescence spectroscopy techniques to confirm these intermediate states in vitro. Finally, we analyzed the deletion of domains to compare our model outputs to previously published results, supporting the presence of interdomain modulation. Overall, our study highlights the significance of interdomain communication within the GRB2 protein and its impact on the formation, stability, and structural plasticity of the protein, which are crucial for its interaction with other proteins in key signaling pathways.

Targeting MET endocytosis or degradation to overcome HGF-induced gefitinib resistance in EGFR-sensitive mutant lung adenocarcinoma

The overexpression of hepatic growth factor(HGF) is one of the important reasons for the development of gefitinib resistance in EGFR-sensitive mutant lung adenocarcinoma cells. Targeting the HGF receptor MET through endocytosis inhibition or degradation induction has been proposed as a potential strategy to overcome this resistance. However, the effectiveness of this approach remains needs to be evaluated. In this study, we observed that MET receptors undergo persistent endocytosis but rarely enter the degradation pathway in HGF-overexpressing cells. We showed that MET endocytosis can be inhibited by using gene silence method or MET inhibitors. CHC or DNM2 gene silence slightly increases the sensitivity of resistant cells to gefitinib without affecting MET activity, while GRB2 gene silence can simultaneously inhibit MET endocytosis and reduce MET activity, resulting in a significant reversal effect of gefitinib resistance. Similarly, MET inhibitors significantly reverse drug resistance, accompanied by simultaneous inhibition of MET endocytosis and activity, highlighting the importance of both endocytosis and activity in HGF-induced gefitinib resistance. Additionally, we demonstrated that promoting MET degradation through deubiquitinase (USP8 or USP32) gene silence is another effective method for reversing drug resistance. Overall, our findings suggest that targeting MET receptor endocytosis and degradation is an attractive strategy for overcoming HGF-induced gefitinib resistance in EGFR-sensitive mutant lung adenocarcinoma.

Assessment of the anti-inflammatory mechanism of quercetin 3,7-dirhamnoside using an integrated pharmacology strategy.

Pouzolzia zeylanica (L.) Benn. is a Chinese herbal medicine widely used for its anti-inflammatory and pus-removal properties. To explore its potential anti-inflammatory mechanism, quercetin 3,7-dirhamnoside (QDR), the main flavonoid component of P. zeylanica (L.) Benn., was extracted and purified. The potential anti-inflammatory targets of QDR were predicted using network analysis. These potential targets were verified using molecular docking, molecular dynamics simulations, and invitro experiments. Consequently, 342 potential anti-inflammatory QDR targets were identified. By analyzing the intersection between the protein-protein interaction and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified several potential protein targets of QDR, including RAC-alpha serine/threonine-protein kinase (AKT1), Ras-related C3 botulinum toxin substrate 1 (RAC1), nitric oxide synthase 3 (NOS3), serine/threonine-protein kinase mTOR (mTOR), epidermal growth factor receptor (EGFR), growth factor receptor-bound protein 2 (GRB2), and endothelin-1 receptor (EDNRA). QDR has anti-inflammatory activity and regulates immune responses and apoptosis through chemokines, Phosphatidylinositol 3-kinase 3(PI3K)/AKT, cAMP, T-cell receptor, and Ras signaling pathways. Molecular docking analysis showed that QDR has good binding abilities with AKT1, mTOR, and NOS3. In addition, molecular dynamics simulations demonstrated that the protein-ligand complex systems formed between QDR and AKT1, mTOR, and NOS3 have high dynamic stability, and their protein-ligand complex systems possess strong binding ability. In RAW264.7 macrophages, QDR significantly inhibited lipopolysaccharides (LPS)-induced inducible nitric oxide synthase expression, nitric oxide (NO) release and the generation of proinflammatory cytokines IL-6, IL-1β, and TNF-α. QDR downregulated the expression of p-AKT1(Ser473)/AKT1 and p-mTOR (Ser2448)/mTOR, and upregulated the expression of NOS3, Rictor, and Raptor. This indicates that the anti-inflammatory mechanisms of QDR involve regulation of AKT1 and mTOR to prevent apoptosis and of NOS3 which leads to the release of endothelial NO. Thus, our study elucidated the potential anti-inflammatory mechanism of QDR, the main flavonoid found in P. zeylanica (L.) Benn.

Grb2 Expression in Acute Spinal Cord Injury After Methylprednisolone Intrathecal Injection in Rats.

This study aims to investigate the effect of methylprednisolone (MP) intrathecal injection on a rat model of acute spinal cord injury (ASCI). Allen's frame was used to establish a rat model of ASCI. MP and normal saline were intrathecally injected to Sprague-Dawley rats at 0, 3, 6, 8, 12, and 24 hours after ASCI, and injured spinal cord tissues were sterilely extracted after 24 hours of treatment. Isobaric tags for relative and absolute quantitation (iTRAQ) were coupled with 2-dimensional liquid chromatography tandem mass spectrometry to separate and identify differentially expressed proteins. The expression of growth factor receptor-bound protein 2 (Grb2) was downregulated in the MP groups at 0 hours (iTRAQ ratio = 0.996), 3 hours (iTRAQ ratio = 0.737), 8 hours (iTRAQ ratio = 0.763), and 24 hours (iTRAQ ratio = 0.908) after injury compared with that in the control groups. No significant difference in Grb2 expression was observed between the control groups at 6 and 12 hours after ASCI. Standardized MP intrathecal injection after ASCI treatment reduces Grb2 activation in a rat ASCI model. Further studies should determine whether or not the same effect can be observed in human ASCIs.

Quantitative proteomic analysis and verification identify global protein profiling dynamics in pig during the estrous cycle.

The current estrus detection method is generally time-consuming and has low accuracy. As such, a deeper understanding of the physiological processes during the estrous cycle accelerates the development of estrus detection efficiency and accuracy. In this study, the label-free acquisition mass spectrometry was used to explore salivary proteome profiles during the estrous cycle (day -3, day 0, day 3, and day 8) in pigs, and the parallel reaction monitoring (PRM) was applied to verify the relative profiles of protein expression. A total of 1,155 proteins were identified in the label-free analysis, of which 115 were identified as differentially expressed proteins (DEPs) among different groups (p ≤ 0.05). Functional annotation revealed that the DEPs were clustered in calcium ion binding, actin cytoskeleton, and lyase activity. PRM verified the relative profiles of protein expression, in which PHB domain-containing protein, growth factor receptor-bound protein 2, elongation factor Tu, carboxypeptidase D, carbonic anhydrase, and trefoil factor 3 were confirmed to be consistent in both label-free and PRM approaches. Comparative proteomic assays on saliva would increase our knowledge of the estrous cycle in sows and provide potential methods for estrus detection.

Optogenetic dissection of RET signaling reveals robust activation of ERK and enhanced filopodia-like protrusions of regenerating axons

RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light–inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

Role of an adaptor protein human germinal center-associated lymphoma (HGAL) in cell signaling and lymphomagenesis

Human germinal center (GC)-associated lymphoma (HGAL) is a multi-domain adaptor protein expressed in GC B lymphocytes, T follicular helper (Tfh) cells and lymphomas derived from these cells. HGAL expression is an independent predictor of longer survival of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin’s lymphoma (HL) patients. HGAL regulates B cell receptor (BCR) signaling and immunological synapse formation by binding to either the downstream effectors [e.g., spleen tyrosine kinase (Syk)] or other signaling regulators [e.g., growth factor receptor-bound protein 2 (Grb2)]. HGAL regulates the cytoskeleton that reshapes B cell morphology during BCR signaling and cell motility by at least two molecular mechanisms: enhanced Ras homolog gene family member A (RhoA) signaling and inhibition of myosin-actin translocation. These effects on the cytoskeleton decrease lymphoma dissemination in animal models and contribute to decreased lymphoma dissemination in patients. The latter may contribute to the association of HGAL protein expression with longer survival of patients with DLBCL and HL tumors. The ability to regulate multiple and distinct functions simultaneously in B cells implies that the HGAL protein level is tightly regulated. It was demonstrated that HGAL can be regulated by PR/SET domain 1 (PRDM1)/B lymphocyte-induced maturation protein-1 (BLIMP1) and interleukin-4 (IL-4) at the transcription level, by microRNA-155 (miR-155) at the post-transcriptional level, and by F-box protein 10 (FBXO10) at the post-translational level. Constitutive enforced expression of HGAL at physiological levels leads to lymphoid hyperplasia and DLBCL in mice. Future studies need to focus on identifying HGAL interactome, dissecting its interaction network, and understanding HGAL spatiotemporal signaling in live cells in physiological conditions. Further, the recent demonstration of HGAL expression in Tfh cells requires the determination of its function in these cells. These studies will contribute to new insights into the biology of these cellular subsets and how immune dysregulation contributes to lymphomagenesis.

Circ_0033596 depletion ameliorates oxidized low-density lipoprotein-induced human umbilical vein endothelial cell damage.

Previous data have shown that circ_0033596 is involved in the pathogenesis of atherosclerosis (AS). The study aims to reveal the detailed mechanism of circ_0033596 in AS. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish an AS cell model. Quantitative real-time polymerase chain reaction and western blot were implemented to detect the expression of circ_0033596, miR-637, growth factor receptor bound protein2 (GRB2), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2). Cell viability, proliferation, apoptosis and tube formation were investigated by cell counting kit-8, EdU assay, flow cytometry and tube formation assay, respectively. The production of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) was evaluated by enzyme-linked immunosorbent assay. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. Dual-luciferase reporter assay, RNA pull-down assay and RIP assay were performed to identify the associations among circ_0033596, miR-637 and GRB2. The expression of circ_0033596 and GRB2 was significantly increased, while miR-637 was decreased in the blood of AS patients and ox-LDL-induced HUVECs compared with controls. Ox-LDL treatment inhibited HUVEC viability, proliferation and angiogenic ability and induced cell apoptosis, inflammation and oxidative stress, while these effects were attenuated after circ_0033596 knockdown. Circ_0033596 interacted with miR-637 and regulated ox-LDL-induced HUVEC damage by targeting miR-637. In addition, GRB2, a target gene of miR-637, participated in ox-LDL-induced HUVEC injury by combining with miR-637. Importantly, circ_0033596 activated GRB2 by interacting with miR-637. Circ_0033596 depletion protected against ox-LDL-induced HUVEC injury by miR-637/GRB2 pathway, providing a therapeutic target for AS.

Structural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling

PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

Regulation of microRNA expression by the adaptor protein GRB2

Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2). The N-terminal SH3 domain of GRB2 is recruited to the PAZ domain of AGO2 forming a ternary complex containing GRB2, AGO2 and DICER1. Using small-RNA sequencing we identified two groups of miRNAs which are regulated by the binding of GRB2. First, mature and precursor transcripts of mir-17~92 and mir-221 miRNAs are enhanced. Second, mature, but not precursor, let-7 family miRNAs are diminished suggesting that GRB2 directly affects loading of these miRNAs. Notably, the resulting loss of let-7 augments expression of oncogenic targets such as RAS. Thus, a new role for GRB2 is established with implications for cancer pathogenesis through regulation of miRNA biogenesis and oncogene expression.

Tauroursodeoxycholic Acid Enhances Osteogenic Differentiation through EGFR/p-Akt/CREB1 Pathway in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are pluripotent stromal cells that are among the most appealing candidates for regenerative medicine and may aid in the repair and regeneration of skeletal disorders through multiple mechanisms, including angiogenesis, differentiation, and response to inflammatory conditions. Tauroursodeoxycholic acid (TUDCA) has recently been used in various cell types as one of these drugs. The mechanism of osteogenic differentiation by TUDCA in hMSCs remains unknown. Cell proliferation was performed by the WST-1 method, and alkaline phosphatase activity and alizarin red-sulfate staining were used to confirm the osteogenic differentiation indicator. Expression of genes related to bone differentiation and specific genes related to signaling pathways was confirmed by quantitative real-time polymerase chain reaction. We found that cell proliferation was higher as the concentration increased, and showed that the induction of osteogenic differentiation was significantly enhanced. We also show that osteogenic differentiation genes were upregulated, with the expression of the epidermal growth factor receptor (EGFR) and cAMP responsive element binding protein 1 (CREB1) being specifically high. To confirm the participation of the EGFR signaling pathway, the osteogenic differentiation index and expression of osteogenic differentiation genes were determined after using an EGFR inhibitor. As a result, EGFR expression was remarkably low, and that of CREB1, cyclin D1, and cyclin E1 was also significantly low. Therefore, we suggest that TUDCA-induced osteogenic differentiation of human MSCs is enhanced through the EGFR/p-Akt/CREB1 pathway.

Cymbaria daurica L.: A Mongolian herbal medicine for treating eczema via natural killer cell-mediated cytotoxicity pathway.

Cymbaria daurica L. (C. daurica) is a perennial herb known commonly as "Xinba" (Chinese) and "Kanba-Arong" (Mongolian). In Mongolia, it is used as a traditional medicine to treat eczema and other skin diseases due to its anti-swelling, anti-inflammatory, anti-hemorrhagic, and anti-itching properties. However, the potential mechanism of action for eczema treatment has not been reported. To investigate the effect of C. daurica on 1-chloro-2,4-dinitrobenzene (DNCB)-induced eczema in rats and the associated action mechanism. Qualitative analysis of C. daurica was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on information obtained from compound identification and relevant literature, the possible targets of C. daurica against eczema were analyzed using network pharmacology and molecular docking methods. The DNCB-induced eczema rat models were treated with different dosages of C. daurica extract (10, 50, and 250mg/mL per day), and the therapeutic effects subsequently evaluated based on the degree of skin inflammation, spleen index, and hematoxylin and eosin staining (H&E staining). Enzyme-linked immunosorbent assay (ELISA), reverse transcription quantitative polymerase chain reaction (RT-qPCR), and western blotting were used to analyze the relevant target effects. The C. daurica mechanism of action on eczema was verified by animal experiments. High-performance liquid chromatography (HPLC) was carried out to determine the content of active ingredients in C. daurica. In addition, the physicochemical properties of the extract were evaluated. Our analysis of the 173 targets included in the protein-protein interaction (PPI) network identified tumor necrosis factor (TNF) and interleukin 2 (IL-2) as key targets involved in the treatment of eczema with C. daurica extract. Furthermore, the 173 targets were associated with the natural killer cell-mediated cytotoxicity pathway. Our results showed that C. daurica significantly reduced IL-2 and TNF-α serum levels in eczema rat models (P<0.0001); thus, playing an important role in the anti-inflammatory response. Furthermore, according to the p-value, RT-qPCR and western blotting showed that the expression of Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1), Vav guanine nucleotide exchange factor (Vav), and growth factor receptor-bound protein 2 (Grb2) changed in the skin of the eczema model rats after treatment with the C. daurica extract. Our study confirms that C. daurica can inhibit SHP-1, Vav, and Grb2 expression; thereby, inhibiting the natural killer cell-mediated cytotoxicity pathway. These results provide insight into the mechanism of C. daurica in treating eczema.

Abstract A025: Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3

Abstract The MAPK/ERK signaling pathway is one of the most heavily mutated in human cancers. In normal cells, the stimulation of receptor tyrosine kinases (e.g. EGFR) on the plasma membrane signals the GTP-loading and activation of RAS through a mechanism catalyzed by the guanine nucleotide exchange factor (GEF) Son of Sevenless 1 (SOS1). RAS activation, in turn, initiates the RAF-MEK-ERK kinase cascade which triggers many downstream signals that can affect gene transcription, protein translation, cell proliferation, and feedback regulation. Several factors regulate SOS1 GEF activity, including two autoinhibitory domains within its own multi-domain architecture. First, the N-terminal domains of SOS1 occlude the allosteric RAS-GTP binding site in the RAS exchanger motif—thereby precluding SOS1 GEF activity—and this inhibition is relieved upon membrane recruitment and PIP2 phospholipid binding. Second, the C-terminal Proline-Rich Region (PRR) of SOS1 also occludes allosteric RAS-GTP binding by an unknown mechanism and is relieved when the growth factor receptor-bound protein 2 (Grb2) binds to motifs in the PRR. While the independent functions of these domains and interactions to inhibition and activation of GEF activity of SOS1 are known, the molecular mechanisms are poorly understood. An additional negative feedback loop involves the phosphorylation of the SOS1 PRR at two sites by the ribosomal protein S6 kinase 1 (RSK1), a substrate of downstream ERK. These modifications create 14-3-3 binding sites on SOS1, where 14-3-3 binding downregulates GEF activity, possibly by obstructing Grb2 association with the SOS1 PRR and membrane localization of SOS1. This work reconstitutes the SOS1:Grb2 and the SOS1:14-3-3 protein complexes in vitro to study the molecular mechanisms underlying SOS1 activity regulation. Citation Format: Orlando E. Martinez, Jawahar Sudhamsu. Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A025.

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2.

The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a 'Src module', an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by inositol hexakisphosphate (IP6) (Wang et al., 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3-bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues - thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.