The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor\'s opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.

Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington\'s disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD.

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.

Protein kinases are molecular machines with rich sequence variation that distinguishes the two main evolutionary branches - tyrosine kinases (TKs) from serine/threonine kinases (STKs). Using a sequence co-variation Potts statistical energy model we previously concluded that TK catalytic domains are more likely than STKs to adopt an inactive conformation with the activation loop in an autoinhibitory folded conformation, due to intrinsic sequence effects. Here we investigate the structural basis for this phenomenon by integrating the sequence-based model with structure-based molecular dynamics (MD) to determine the effects of mutations on the free energy difference between active and inactive conformations, using a thermodynamic cycle involving many (n = 108) protein-mutation free energy perturbation (FEP) simulations in the active and inactive conformations. The sequence and structure-based results are consistent and support the hypothesis that the inactive conformation DFG-out Activation Loop Folded, is a functional regulatory state that has been stabilized in TKs relative to STKs over the course of their evolution via the accumulation of residue substitutions in the activation loop and catalytic loop that facilitate distinct substrate binding modes in trans and additional modes of regulation in cis for TKs.

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a \'timely two-ness\' that allows cell division to occur in absence of a SAC-dependent mitotic delay.

暂无摘要。

The splicing factor RNA-binding motif protein 10 (RBM10) is frequently mutated in lung adenocarcinoma (LUAD) (9-25%). Most RBM10 cancer mutations are loss-of-function, correlating with increased tumorigenesis and limiting the efficacy of current LUAD targeted therapies. Remarkably, therapeutic strategies leveraging RBM10 deficiency remain unexplored. Here, we conduct a CRISPR-Cas9 synthetic lethality (SL) screen and identify ~60 RBM10 SL genes, including WEE1 kinase. WEE1 inhibition sensitizes RBM10-deficient LUAD cells in-vitro and in-vivo. Mechanistically, we identify a splicing-independent role of RBM10 in regulating DNA replication fork progression and replication stress response, which underpins RBM10-WEE1 SL. Additionally, RBM10 interacts with active DNA replication forks, relying on DNA Primase Subunit 1 (PRIM1) that synthesizes Okazaki RNA primers. Functionally, we demonstrate that RBM10 serves as an anchor for recruiting Histone Deacetylase 1 (HDAC1) to facilitate H4K16 deacetylation and R-loop homeostasis to maintain replication fork stability. Collectively, our data reveal a role of RBM10 in fine-tuning DNA replication and provide therapeutic arsenal for targeting RBM10-deficient tumors.

The TEC (tyrosine kinase expressed in hepatocellular carcinoma) family kinases, an important class of protein kinases, play key roles in cell signaling and immune regulation. In this review, the association between the pathogenesis of alopecia areata and the structure and regulatory mechanisms of TEC family kinases in normal physiological processes, is explored. Furthermore, the potential clinical applications of TEC kinases and their inhibitors are highlighted by summarizing current insights into their mechanisms of action, clinical applications, and recent advancements in TEC kinase inhibitor research.

Malignant pleural effusion (MPE) from patients with advanced non-small-cell lung cancer (NSCLC) has been proven valuable for molecular analysis; however, simultaneous detection of driver fusions in MPE is still challenging. In this study, we investigated the Idylla™ GeneFusion Panel, a stand-alone test in tissue samples, in the evaluation of ALK, ROS1, RET and MET ex14 skipping mutations in MPE and compared its performance with routine reference methods (Real-time-based and Next-generation Sequencing-NGS). The inclusion criteria for sample selection were as follows: advanced NSCLC harboring ALK, ROS1, RET fusions or MET exon-skipping alterations and the availability of MPE collected at diagnosis or disease progression. Molecular alterations have been investigated on tissue by fluorescence in situ hybridization (FISH) or Real-time PCR or NGS. For molecular profiling with the Idylla™ GeneFusion, 200 µL of MPE supernatants combined with 50 µL of RNA Later solution were loaded into the Idylla™ cartridge without cfRNA extraction. The Idylla™ GeneFusion Assay performed on MPEs was able to confirm molecular profile, previously diagnosed with conventional methods, in all cases. Our data confirm that MPE are suitable material for investigating fusion alterations. The Idylla™ GeneFusion, although indicated for investigation of tissue samples, offers the possibility of performing a molecular characterization of supernatants without undertaking the entire cfRNA extraction procedure providing a rapid and reliable strategy for the detection of actionable genetic alterations.

OBJECTIVE: Acute-on-chronic liver failure (ACLF) is a syndrome characterized by a high short-term mortality rate, and effective interventions are still lacking. This study aims to investigate whether the small molecule baicalein can mitigate ACLF and elucidate the molecular mechanisms.
METHODS: The ACLF mouse model was induced through chronic liver injury using carbon tetrachloride, followed by acute inflammation induction with lipopolysaccharide (LPS). Baicalein was administered through intraperitoneal injection to explore its therapeutic effects. In vitro experiments utilized the iBMDM macrophage cell line to investigate the underlying mechanisms. Peripheral blood was collected from clinical ACLF patients for validation.
RESULTS: In the LPS-induced ACLF mouse model, baicalein demonstrated a significant reduction in acute inflammation and liver damage, as evidenced by histopathological evaluation, liver function analysis, and inflammatory marker measurements. Transcriptomic analysis, coupled with molecular biology experiments, uncovered that baicalein exerts its effects in ACLF by activating the TrKB-CREB1 signaling axis to upregulate the surface expression of the TREM2 receptor on macrophages. This promotes M2 macrophage polarization and activates efferocytosis, thereby inhibiting inflammation and alleviating liver damage. Furthermore, we observed a substantial negative correlation between postoperative peripheral blood plasma soluble TREM2 (sTREM2) levels and inflammation, as well as adverse outcomes in clinical ACLF patients.
CONCLUSIONS: Baicalein plays a protective role in ACLF by enhancing the surface expression of the TREM2 receptor on macrophages, leading to the suppression of inflammation, mitigation of liver damage, and a reduction in mortality. Additionally, plasma sTREM2 emerges as a critical indicator for predicting adverse outcomes in ACLF patients.