Search Results (20)

Nestin is a type VI intermediate filament protein and a well-known neural stem cell marker. It is also expressed in high-grade cancer cells, forming copolymerized filaments with vimentin. We previously showed that nestin inhibits the binding of vimentin’s tail domain to actin filaments (AFs) by steric hindrance through its large nestin tail domain (NTD), thereby increasing three-dimensional cytoskeleton network mobility, enhancing cell flexibility, and promoting cancer progression. Further, we found that nestin itself stably binds to AFs via the NTD. We therefore hypothesized that the NTD may form a flexible cytoskeletal structure by extending with weak force. In vitro tensile tests using atomic force microscopy were performed to assess the mechanical properties of NTDs. The C-terminus of the NTD bound AFs by bringing the AFM tip modified with the NTD into contact with the AFs on the substrate. NTDs were elongated to approximately 80% of their maximum length at weak forces < 150 pN. Repeated tensile tests revealed that the NTD refolded quickly and behaved like a soft elastic material. We speculate that nestin stably binds AFs, and the NTD extends with weak force, contracting quickly upon load release. Thereby, nestin would absorb mechanical load and maintain cytoskeletal integrity. Full article

The TaqIA polymorphism is a marker of both the Ankyrin Repeat and Kinase Domain containing I gene (ANKK1) encoding a RIP-kinase, and the DRD2 gene for the dopamine receptor D2. Despite a large number of studies of TaqIA in addictions and other psychiatric disorders, there is difficulty in interpreting this genetic phenomenon due to the lack of knowledge about ANKK1 function. In SH-SY5Y neuroblastoma models, we show that ANKK1 interacts with the synapse protein FERM ARH/RhoGEF and Pleckstrin Domain 1 (FARP1), which is a guanine nucleotide exchange factor (GEF) of the RhoGTPases RAC1 and RhoA. ANKK1–FARP1 colocalized in F-ACTIN-rich structures for neuronal maturation and migration, and both proteins activate the Wnt/PCP pathway. ANKK1, but not FARP1, promotes neuritogenesis, and both proteins are involved in neuritic spine outgrowth. Notably, the knockdown of ANKK1 or FARP1 affects RhoGTPases expression and neural differentiation. Additionally, ANKK1 binds WGEF, another GEF of Wnt/PCP, regulating its interaction with RhoA. During neuronal differentiation, ANKK1–WGEF interaction is downregulated, while ANKK1–FARP1 interaction is increased, suggesting that ANKK1 recruits Wnt/PCP components for bidirectional control of F-ACTIN assembly. Our results suggest a brain structural basis in TaqIA-associated phenotypes. Full article

Human cancers express altered levels of actin-binding cytoskeletal filamin A (FLNA) protein. FLNA in mammals consists of an actin-binding domain at its N-terminus that is followed by 24 immunoglobulin-like repeat modules interrupted by two hinge regions between repeats 15–16 and 23–24. Cleavage of these hinge regions produces a naturally occurring C-terminal 90 kDa fragment of FLNA (FLNA^CT) that physically interacts with multiple proteins with diverse functions. This cleavage leads to actin cytoskeleton remodeling, which in turn contributes to cellular signaling, nucleocytoplasmic shuttling of transcriptional factors and nuclear receptors, and regulation of their transcriptional activities that are important for initiation and progression of cancers. Therefore, recent studies have proposed blocking FLNA cleavage as a means of cancer therapy. Here, we update how FLNA cleavage has been targeted by different approaches and their potential implications for future treatment of human cancers. Full article

Hepatocellular carcinoma (HCC) is a prevalent malignant digestive tumor. Numerous genetic mutations have been documented in HCC, yet the clinical significance of these mutations remains largely unexplored. The objective of this study is to ascertain the clinical value and biological effects of xin actin binding repeat containing 2 (XIRP2) mutation in HCC. The gene mutation landscape of HCC was examined using data from the Cancer Genome Atlas and the International Cancer Genome Consortium databases. The prognostic significance of the XIRP2 mutation was assessed through KM plot analysis. The association between drug sensitivity and the XIRP2 mutation was investigated using the TIDE algorithm and CCK-8 experiments. The biological effects of the XIRP2 mutation were evaluated through qRT-PCR, protein stability experiments, and relevant biological experiments. The XIRP2 mutation is one of the high-frequency mutations in HCC, and is associated with poor prognosis. A total of 72 differentially expressed genes (DEGs) were observed in HCC tissues with the XIRP2 mutation as compared to those with the XIRP2 wildtype, and these DEGs were closely related to ion metabolic processes. The XIRP2 mutation was linked to alterations in the sensitivity of fludarabine, oxaliplatin, WEHI-539, and LCL-161. CCK-8 assays demonstrated that HCC cells carrying the XIRP2 mutation exhibited increased resistance to fludarabine and oxaliplatin, but enhanced sensitivity to WEHI-539 and LCL-161 as compared with those HCC cells with the XIRP2 wildtype. The XIRP2 mutation was found to have no impact on the mRNA levels of XIRP2 in tissues and cells, but it did enhance the stability of the XIRP2 protein. Mechanically, the inhibition of XIRP2 resulted in a significant increase in sensitivity to oxaliplatin through an elevation in zinc ions and a calcium ion overload. In conclusion, the XIRP2 mutation holds potential as a biomarker for predicting the prognosis and drug sensitivity of HCC and serves as a therapeutic target to enhance the efficacy of oxaliplatin. Full article

Idiopathic pulmonary fibrosis has poor clinical outcomes despite antifibrotic treatment. The nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome and endothelial-to-mesenchymal transition (EndoMT) were shown to be involved in the pathogenesis of pulmonary fibrosis. However, the detailed mechanism is unknown. Our study aimed to investigate the role of the NLRP3 inflammasome in the regulation of EndoMT in pulmonary fibrosis. The inhibition of the NLRP3 inflammasome via a caspase-1 inhibitor, Ac-YVAD-cmk (YVAD), was intraperitoneally administered to male C57BL/6 mice (8–12 weeks old) one hour before bleomycin intratracheal injection (1.5 U/kg). Immunohistochemical staining, Masson’s trichrome staining, enzyme-linked immunosorbent assay, immunofluorescence, and Western blotting were used to assess the activity of the NLRP3 inflammasome and EndoMT in lung samples from mice. Human pulmonary microvascular endothelial cells (HPMECs) were used as a model of EndoMT in vitro with YVAD and bleomycin stimulation. We observed the activation of the NLRP3 inflammasome and EndoMT (decreased vascular endothelial cadherin with increased alpha-smooth muscle actin and vimentin) in the lung samples after bleomycin. However, inhibition of the NLRP3 inflammasome significantly reduces EndoMT via inhibiting focal adhesion kinase (FAK). In vitro studies also confirmed these findings. In conclusion, NLRP3 inflammasome inhibition could reduce lung inflammation and fibrosis via the regulation of EndoMT by the FAK pathway. Full article

Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell–cell adhesion. Full article

Genetic and environmental factors are associated with developing and progressing duodenal ulcer (DU) risk. However, the exact nature of the disease pathophysiology and the single nucleotide polymorphism (SNP)—lifestyle interaction has yet to be determined. The purpose of the present study was to examine the SNPs linked to DU risk and their interaction with lifestyles and diets in a large hospital-based cohort of Asians. Based on an earlier diagnosis, the participants were divided into the DU (case; n = 1088) and non-DU (control, n = 56,713) groups. The SNP associated with DU risk were obtained from a genome-wide association study (GWAS), and those promoted genetic impact with SNP–SNP interactions were identified with generalized multifactor dimensionality reduction analysis. The interaction between polygenic risk score (PRS) calculated from the selected genetic variants and nutrient were examined. They were related to actin modification, immune response, and cell migration by modulating leucine-rich repeats (LRR) domain binding, Shaffer interferon regulatory factor 4 (IRF4) targets in myeloma vs. mature B lymphocyte, and Reactome runt-related transcription factor 3 (RUNX3). Among the selected SNPs, rs11230563 (R225W) showed missense mutation and low binding affinity with different food components in the autodock analysis. Glycyrrhizin, physalin B, janthitrem F, and casuarinin lowered it in only wild CD6 protein but not in mutated CD6. Plastoquinone 8, solamargine, saponin D, and matesaponin 2 decreased energy binding affinity in mutated CD6 proteins. The PRS of the 5-SNP and 6-SNP models exhibited a positive association with DU risk (OR = 3.14). The PRS of the 5-SNP PRS model interacted with irregular eating habits and smoking status. In participants with irregular eating habits or smokers, DU incidence was much higher in the participants with high PRS than in those with low PRS. In conclusion, the genetic impact of DU risk was mainly in regulating immunity, inflammation, and actin modification. Adults who are genetically susceptible to DU need to eat regularly and to be non-smokers. The results could be applied to personalize nutrition. Full article

Filamin protein is characterized by an N-terminal actin-binding domain that is followed by 24 Ig (immunoglobulin)-like repeats, which act as hubs for interactions with a variety of proteins. In humans, this family has been found to be involved in cancer cell invasion and metastasis and can be involved in a variety of growth signal transduction processes, but it is less studied in plants. Therefore, in this study, 54 Filamin gene family members from 23 plant species were investigated and divided into two subfamilies: FLMN and GEX2. Subcellular localization showed that most of the Filamin gene family members were located in the cell membrane. A total of 47 Filamin gene pairs were identified, most of which were whole-genome copies. Through the analyses of cis-acting elements, expression patterns and quantitative fluorescence, it was found that GH_ A02G0519 and GH_ D02G0539 are mainly expressed in the reproductive organs of upland cotton, and their interacting proteins are also related to the fertilization process, whereas GH_A02G0216 and GH_D02G0235 were related to stress. Thus, it is speculated that two genes of the GEX2 subfamily (GH_A02G0519 and GH_D02G0539) may be involved in the reproductive development of cotton and may affect the fertilization process of cotton. This study provides a theoretical basis for the further study of the cotton Filamin gene family. Full article

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid–liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the ARPIN locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence. Full article

Vitamin D, unlike the micronutrients, vitamins A, E, and K, is largely obtained not from food, but by the action of solar ultraviolet (UV) light on its precursor, 7-dehydrocholesterol, in skin. With the decline in UV light intensity in winter, most skin production of vitamin D occurs in summer. Since no defined storage organ or tissue has been found for vitamin D, it has been assumed that an adequate vitamin D status in winter can only be maintained by oral supplementation. Skeletal muscle cells have now been shown to incorporate the vitamin D-binding protein (DBP) from blood into the cell cytoplasm where it binds to cytoplasmic actin. This intracellular DBP provides an array of specific binding sites for 25-hydroxyvitamin D (25(OH)D), which diffuses into the cell from the extracellular fluid. When intracellular DBP undergoes proteolytic breakdown, the bound 25(OH)D is then released and diffuses back into the blood. This uptake and release of 25(OH)D by muscle accounts for the very long half-life of this metabolite in the circulation. Since 25(OH)D concentration in the blood declines in winter, its cycling in and out of muscle cells appears to be upregulated. Parathyroid hormone is the most likely factor enhancing the repeated cycling of 25(OH)D between skeletal muscle and blood. This mechanism appears to have evolved to maintain an adequate vitamin D status in winter. Full article

Since its belated discovery, our understanding of the giant protein titin has grown exponentially from its humble beginning as a sarcomeric scaffold to recent recognition of its critical mechanical and signaling functions in active muscle. One uniquely useful model to unravel titin’s functions, muscular dystrophy with myositis (mdm), arose spontaneously in mice as a transposon-like LINE repeat insertion that results in a small deletion in the N2A region of titin. This small deletion profoundly affects hypertrophic signaling and muscle mechanics, thereby providing insights into the function of this specific region and the consequences of its dysfunction. The impact of this mutation is profound, affecting diverse aspects of the phenotype including muscle mechanics, developmental hypertrophy, and thermoregulation. In this review, we explore accumulating evidence that points to the N2A region of titin as a dynamic “switch” that is critical for both mechanical and signaling functions in skeletal muscle. Calcium-dependent binding of N2A titin to actin filaments triggers a cascade of changes in titin that affect mechanical properties such as elastic energy storage and return, as well as hypertrophic signaling. The mdm phenotype also points to the existence of as yet unidentified signaling pathways for muscle hypertrophy and thermoregulation, likely involving titin’s PEVK region as well as the N2A signalosome. Full article

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction. Full article

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is a protein that belongs to the Arf GAP (GTPase activating protein) protein family. These proteins act as GTPase switches for Arfs, which are Ras superfamily members, being therefore involved in signaling regulation. Arf GAP proteins have been shown to participate in several cellular functions including membrane trafficking and actin cytoskeleton remodeling. AGAP2 is a multi-tasking Arf GAP that also presents GTPase activity and is involved in several signaling pathways related with apoptosis, cell survival, migration, and receptor trafficking. The increase of AGAP2 levels is associated with pathologies as cancer and fibrosis. Transforming growth factor beta-1 (TGF-β1) is the most potent pro-fibrotic cytokine identified to date, currently accepted as the principal mediator of the fibrotic response in liver, lung, and kidney. Recent literature has described that the expression of AGAP2 modulates some of the pro-fibrotic effects described for TGF-β1 in the liver. The present review is focused on the interrelated molecular effects between AGAP2 and TGFβ1 expression, presenting AGAP2 as a new player in the signaling of this pro-fibrotic cytokine, thereby contributing to the progression of hepatic fibrosis. Full article

Influenza A virus (IAV) exploits host factors to multiply and cause disease. An in-depth knowledge of this interaction of IAV with the host will aid the development of anti-IAV intervention strategies. Previously, we demonstrated that host cortactin, an actin filament-binding protein promotes IAV infection, but undergoes degradation via a lysosome-associated apoptotic pathway during the late stages of IAV infection. Next, we wanted to further understand the mechanisms and significance of this phenomenon. By using the RNA interference screens and site-directed mutagenesis followed by western blotting, we found that lysosome protease, cathepsin C is involved in cortactin degradation in human cells infected with IAV. Furthermore, executioner apoptotic caspase, caspase-3 not caspase-6 or caspase-7 is involved in cortactin degradation during IAV infection, and caspase-3 cleavage site is located in the first actin-binding repeat of cortactin polypeptide. Finally, when expressed ectopically, the cleavage-resistant cortactin mutants decreased the amount of IAV progeny released from infected cells that was enhanced by the cleavage-sensitive cortactin wild type. These data strengthen the hypothesis proposed earlier that host cortactin plays an inhibitory role during the late stages of IAV infection, and IAV is facilitating its degradation to undermine such function. Full article

The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells. Full article