Deciphering the Cofilin Oligomers via Intermolecular Disulfide Bond Formation: A Coarse-Grained Molecular Dynamics Approach to Understanding Cofilin's Regulation on Actin Filaments.

Cofilin, a key actin-binding protein, orchestrates the dynamics of the actomyosin network through its actin-severing activity and by promoting the recycling of actin monomers. Recent experiments suggest that cofilin forms functionally distinct oligomers via thiol post-translational modifications (PTMs) that promote actin nucleation and assembly. Despite these advances, the structural conformations of cofilin oligomers that modulate actin activity remain elusive because there are combinatorial ways to oxidize thiols in cysteines to form disulfide bonds rapidly. This study employs molecular dynamics simulations to investigate human cofilin 1 as a case study for exploring cofilin dimers via disulfide bond formation. Utilizing a biasing scheme in simulations, we focus on analyzing dimer conformations conducive to disulfide bond formation. Additionally, we explore potential PTMs arising from the examined conformational ensemble. Using the free energy profiling, our simulations unveil a range of probable cofilin dimer structures not represented in current Protein Data Bank entries. These candidate dimers are characterized by their distinct population distributions and relative free energies. Of particular note is a dimer featuring an interface between cysteines 139 and 147 residues, which demonstrates stable free energy characteristics and intriguingly symmetrical geometry. In contrast, the experimentally proposed dimer structure exhibits a less stable free energy profile. We also evaluate frustration quantification based on the energy landscape theory in the protein-protein interactions at the dimer interfaces. Notably, the 39-39 dimer configuration emerges as a promising candidate for forming cofilin tetramers, as substantiated by frustration analysis. Additionally, docking simulations with actin filaments further evaluate the stability of these cofilin dimer-actin complexes. Our findings thus offer a computational framework for understanding the role of thiol PTM of cofilin proteins in regulating oligomerization, and the subsequent cofilin-mediated actin dynamics in the actomyosin network.

The amoeboid migration of monocytes in confining channels requires the local remodeling of the cortical actin cytoskeleton by cofilin-1.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.

Identification of an immune-related gene signature for predicting prognosis and immunotherapy efficacy in liver cancer via cell-cell communication.

BACKGROUND: Liver cancer is one of the deadliest malignant tumors worldwide. Immunotherapy has provided hope to patients with advanced liver cancer, but only a small fraction of patients benefit from this treatment due to individual differences. Identifying immune-related gene signatures in liver cancer patients not only aids physicians in cancer diagnosis but also offers personalized treatment strategies, thereby improving patient survival rates. Although several methods have been developed to predict the prognosis and immunotherapeutic efficacy in patients with liver cancer, the impact of cell-cell interactions in the tumor microenvironment has not been adequately considered. AIM: To identify immune-related gene signals for predicting liver cancer prognosis and immunotherapy efficacy. METHODS: Cell grouping and cell-cell communication analysis were performed on single-cell RNA-sequencing data to identify highly active cell groups in immune-related pathways. Highly active immune cells were identified by intersecting the highly active cell groups with B cells and T cells. The significantly differentially expressed genes between highly active immune cells and other cells were subsequently selected as features, and a least absolute shrinkage and selection operator (LASSO) regression model was constructed to screen for diagnostic-related features. Fourteen genes that were selected more than 5 times in 10 LASSO regression experiments were included in a multivariable Cox regression model. Finally, 3 genes (stathmin 1, cofilin 1, and C-C chemokine ligand 5) significantly associated with survival were identified and used to construct an immune-related gene signature. RESULTS: The immune-related gene signature composed of stathmin 1, cofilin 1, and C-C chemokine ligand 5 was identified through cell-cell communication. The effectiveness of the identified gene signature was validated based on experimental results of predictive immunotherapy response, tumor mutation burden analysis, immune cell infiltration analysis, survival analysis, and expression analysis. CONCLUSION: The findings suggest that the identified gene signature may contribute to a deeper understanding of the activity patterns of immune cells in the liver tumor microenvironment, providing insights for personalized treatment strategies.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail.

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-µm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.

Neuronal CFL1 upregulation in head and neck squamous cell carcinoma enhances tumor-nerve crosstalk and promotes tumor growth.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, marked by a pronounced nerve density within the tumor microenvironment and a high rate of perineural invasion (PNI). Growing evidence suggests that the nervous system plays a vital role in HNSCC progression. Yet, the mechanisms governing cancer-nerve interactions remain largely elusive. Our research revealed that cofilin-1 (CFL1) is significantly overexpressed in HNSCC and correlates with both PNI and unfavorable prognosis. Utilizing multiplex fluorescent immunohistochemistry, we have localized CFL1 chiefly to the nerves adjacent to tumor sites. Significantly, it is the elevated expression of CFL1 in neuronal structures, rather than in the tumor cells, that aligns with diminished patient survival rates. We observed that HNSCC cells induced the expression of neuronal CFL1 and that the conditional knockout of neuronal CFL1 impedes tumor-nerve interactions. Both Gene Ontology functional enrichment analyses and Gene Set Enrichment Analysis demonstrate that CFL1 expression in HNSCC is associated with specific biological processes, including "RIBOSOME," "PROTEASOME," and "cadherin binding." In summary, HNSCC promotes the expression of CFL1 in nerves, which is essential for cancer-nerve interactions. The neuronal CFL1 is associated with PNI and may be a potential molecular prognostic marker of poor survival in HNSCC.

CFL1 restores the migratory capacity of bone marrow mesenchymal stem cells in primary Sjögren's syndrome by regulating CCR1 expression.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease. There is no relevant research on whether the migratory ability of bone marrow mesenchymal stem cells (BM-MSC) is impaired in patients with pSS (pSS-BMMSC). METHODS: Trajectories and velocities of BM-MSC were analyzed. Transwell migration assay and wound healing assay were used to investigate the migratory capacity of BM-MSC. The proliferative capacity of BM-MSC was evaluated by EDU and CCK8 assay. RNA-seq analysis was then performed to identify the underlying mechanism of lentivirus-mediated cofilin-1 overexpression BM-MSC (BMMSCCFL1). The therapeutic efficacy of BMMSCCFL1 was evaluated in NOD mice. RESULTS: The migratory capacity of pSS-BMMSC was significantly reduced compared to normal volunteers (HC-BMMSC). The expression of the motility-related gene CFL1 was decreased in pSS-BMMSC. Lentivirus-mediated CFL1 overexpression of pSS-BMMSC promoted the migration capacity of pSS-BMMSC. Furthermore, RNA-seq revealed that CCR1 was the downstream target gene of CFL1. To further elucidate the mechanism of CFL1 in regulating BM-MSC migration and proliferation via the CCL5/CCR1 axis, we performed a rescue experiment using BX431 (a CCR1-specific inhibitor) to inhibit CCR1. The results showed that CCR1 inhibitors suppressed the migration and proliferation capacity of MSC induced by CFL1. CONCLUSION: The pSS-BMMSC leads to impaired migration and proliferation, and overexpression of CFL1 can rescue the functional deficiency and alleviate disease symptoms in NOD mice. Mechanically, CFL1 can regulate the expression level of the downstream CCL5/CCR1 axis to enhance the migration and proliferation of BM-MSC.

Remodeling Serine Synthesis and Metabolism via Nanoparticles (NPs)-Mediated CFL1 Silencing to Enhance the Sensitivity of Hepatocellular Carcinoma to Sorafenib.

Tyrosine kinase inhibitors represented by sorafenib are the first-line treatment for hepatocellular carcinoma (HCC), but the low response rate of HCC patient has become a clinical pain-point. Emerging evidences have revealed that metabolic reprogramming plays an important role in regulating the sensitivity of tumor cells to various chemotherapeutics including sorafenib. However, the underlying mechanisms are very complex and are not fully elucidated. By comparing the transcriptome sequencing data of sorafenib-sensitive and -insensitive HCC patients, it is revealed that cofilin 1 (CFL1) is highly expressed in the tumor tissues of sorafenib-insensitive HCC patients and closely correlated with their poor prognosis. Mechanically, CFL1 can promote phosphoglycerate dehydrogenase transcription and enhance serine synthesis and metabolism to accelerate the production of antioxidants for scavenging the excessive reactive oxygen species induced by sorafenib, thereby impairing the sorafenib sensitivity of HCC. To translate this finding and consider the severe side effects of sorafenib, a reduction-responsive nanoplatform for systemic co-delivery of CFL1 siRNA (siCFL1) and sorafenib is further developed, and its high efficacy in inhibiting HCC tumor growth without apparent toxicity is demonstrated. These results indicate that nanoparticles-mediated co-delivery of siCFL1 and sorafenib can be a new strategy for the treatment of advanced HCC.

Cofilin promotes tau pathology in Alzheimer's disease.

The molecular mechanisms mediating the aggregation and transmission of tau in AD remain unclear. Here, we show that the actin-binding protein cofilin is cleaved by a cysteine protease asparagine endopeptidase (AEP) at N138 in the brains of patients with AD. The AEP-generated cofilin 1-138 fragment interacts with tau and promotes its aggregation. The mixed fibrils consisting of cofilin 1-138 and tau are more pathogenic to cells than pure tau fibrils. Furthermore, overexpression of cofilin 1-138 in the brain facilitates the propagation of pathological tau aggregates and promotes AD-like cognitive impairments in tau P301S mice. However, mice infected with adeno-associated viruses (AAVs) encoding an AEP-uncleavable cofilin mutant show attenuated tau pathology and cognitive impairments compared with mice injected with AAVs encoding wild-type cofilin. Together, these observations support the role of the cofilin 1-138 fragment in the aggregation and transmission of tau pathology during the onset and progression of AD.

Cofilin 1 Is Dynamically Modulated During Postnatal Development and by Visual Input in the Mouse Visual Cortex.

Cofilin 1 is an actin depolymerizing protein playing a fundamental role in the turnover of actin filaments specifically in dendritic spines, where it has been associated with structural and functional plasticity processes. Using a differential proteomic approach, we recently identified cofilin 1 as a potential candidate for controlling plasticity levels in the mouse visual cortex. Here, we focus on analyzing the expression of cofilin 1 and of its serine-3 phosphorylated inactive form in the mouse visual cortex during postnatal development and its modulation by visual input. Western blot experiments showed that cofilin 1 decreases from the critical period to the adult stage, in correlation with the decreasing level of cortical plasticity, and that monocular deprivation increases its expression in the cortex contralateral to the deprived eye during the critical period but not in the adult stage. By immunohistochemistry, we identified that the phospho-cofilin 1 immunopositive signal is homogeneously expressed along the different layers of the mouse visual cortex and that it increases during postnatal development. Furthermore, monocular deprivation increases the phospho-cofilin 1 signal in the contralateral cortex to the deprived eye during the critical period but not in the adult stage. Altogether, these results suggest that cofilin 1 and its modification by phosphorylation are relevant players in the processes controlling experience-dependent plasticity in the mouse visual cortex.

Celastrus orbiculatus Thunb. extract inhibits EMT and metastasis of gastric cancer by regulating actin cytoskeleton remodeling.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine herb Celastrus orbiculatus Thunb. is an important folk medicinal plant in China that has been used as an anti-inflammatory, antitumor, and analgesic in various diseases. Recent years, many studies have reported the significant effects of Celastrus orbiculatus Thunb. extract (COE) on gastric cancer. However, the specific mechanism by which COE regulates gastric cancer cytoskeleton remodeling and thus inhibits EMT has not yet been reported. AIM OF STUDY: To study the effect and mechanism of COE in inhibiting the epithelial-mesenchymal transition (EMT) and metastasis of gastric cancer cells, laying an experimental foundation for the clinical application and further development of COE. METHODS: The high-content cell dynamic tracking system was used to continuously track the trajectory of cell movement in real time. Through the high-content data, the average movement distance and movement speed of the cells are calculated. Additionally, the dynamic images of the cell movement in the high-content imaging system are derived to analyze the impact of COE on the movement of gastric cancer cells. Cytoskeleton staining experiment was performed to detect the effect of COE on the assembly of gastric cancer cell cytoskeleton proteins. Western blot was employed to detect the changes of EMT and metastasis-related proteins in the gastric cancer cells treated by COE. The effect of COE on the key regulatory protein Cofilin-1 (CFL1) of cell movement was examined by Western blot and protein degradation experiment. The effect of COE on EMT and metastasis of the gastric cancer cells lacking CFL1 was assessed by a transwell assay. The in vivo inhibitory effect of COE on EMT and metastasis of gastric cancer was determined by the animal living image system. IHC assays were used to detect the levels of EMT-related proteins in COE reversal in vivo. RESULT: The results showed that the movement distance and average movement speed of gastric cancer cells after COE treatment were significantly lower than those of the control group. Cytoskeleton staining experiments revealed that COE can significantly change the distribution of skeletal proteins in gastric cancer cells. Additionally, COE treatment significantly reduced the expression of Matrix metalloproteinases (MMP-2, MMP-9) and other proteins. Furthermore, COE can significantly accelerate the degradation of CFL1 protein, and both COE treatment and CFL1 deletion can significantly inhibit EMT and metastasis of gastric cancer cells. Lastly, the number of peritoneal metastases of gastric cancer cells was significantly reduced in animals after COE treatment. COE can reverse the levels of EMT-related proteins while reducing the expression levels of CFL1 protein in vivo. CONCLUSION: COE can significantly inhibit EMT and metastasis of gastric cancer cells in vivo and in vitro. This effect may be achieved by reducing the stability of CFL1 and inhibiting the assembly of actin in gastric cancer cells.

The phosphorylation level of Cofilin-1 is related to the pathological subtypes of gastric cancer.

The purpose of the study was to explore the relationship between multiple proteins belonging to the LIMK/Cofilin pathway, including LIMK1, LIMK2, Cofilin-1, and p-Cofilin-1 and clinical features of gastric cancer (GC) patients, including overall survival, TNM stages, and pathological subtypes. The expression of LIMK1, LIMK2, Cofilin-1 and p-Cofilin-1 in the GC tissues and adjacent normal stomach tissues from 141 patients were detected using immunohistochemistry (IHC) staining. Wilcoxon rank-sum test and Spearman rank correlation coefficients were used to measure the relationship between different TNM stages, pathological types, and selected parameters. OS was estimated using the Kaplan-Meier method and survival curves were compared using the log-rank test. Our results showed that, compared to those in the adjacent normal stomach tissues, LIMK1, LIMK2 and Cofilin-1 were up-regulated while p-Cofilin-1 was down-regulated in the GC tissues. LIMK1 level was positively correlated to the TNM stages of GC. According to the published dataset, the expression levels of both LIMK1 and LIMK2 were correlated to the overall survival time of GC patients. The level of Cofilin-1 was significantly different between GCs of different TNM stages. Moreover, most importantly, this is the first study to reveal that the level of Cofilin-1 is higher, and the level of p-Cofilin-1 is lower in the diffuse type of GC compared to that in intestinal type. Taken together, our study demonstrated that multiple factors in LIMK/Cofilin pathway including LIMK1, LIMK2, Cofilin-1, and p-Cofilin-1 were associated with the clinical and pathological features of GC, which is potentially helpful for the diagnosis and treatment of GC.

Cofilin-1 and profilin-1 expression in lung microvascular endothelial cells exposed to titanium dioxide nanoparticles.

BACKGROUND: Air pollutants exacerbate chronic airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). However, the underlying mechanisms are yet to be determined. While a number of studies have reported adverse effects of nanoparticles on humans, little is known about their effects on the respiratory system. OBJECTIVES: To examine the protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to titanium dioxide (TiO2) nanoparticles, a common air pollutant. MATERIAL AND METHODS: A proteomics approach using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was used to determine the differences in protein expression at 8 h and 24 h, following the treatment of HMVEC-L with 20-µM or 40-µM TiO2 nanoparticles. RESULTS: Human lung microvascular endothelial cells treated with 20-µM TiO2 nanoparticles showed alterations of 7 protein spots, including molecules related to calcium regulation, transport, cytoskeleton, and muscle contraction. The treatment of HMVEC-L with 40-µM TiO2 nanoparticles resulted in alterations of 4 protein spots, with molecular functions related to the cytoskeleton, myosin regulation, actin modulation, as well as guanosine diphosphate (GDP) and guanosine triphosphate (GTP) regulation. To validate these results, immunohistochemical staining and western blotting analyses were performed on lung tissues collected from mice exposed to TiO2 nanoparticles. Cofilin-1 and profilin-1 were expressed in the endothelium, epithelium and inflammatory cells, and decreased in lung tissues of TiO2 nanoparticle-exposed mice compared to sham-treated controls. CONCLUSIONS: These results suggest that some of the differentially expressed proteins may play important roles in airway diseases caused by TiO2 nanoparticle exposure.

Magic angle spinning NMR structure of human cofilin-2 assembled on actin filaments reveals isoform-specific conformation and binding mode.

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.

Estradiol inhibits HIV-1BaL infection and induces CFL1 expression in peripheral blood mononuclear cells and endocervical mucosa.

An inhibitory effect of estradiol (E2) on HIV-1 infection was suggested by several reports. We previously identified increased gene expression of actin-binding protein cofilin 1 (CFL1) in endocervix in the E2-dominated proliferative phase of the menstrual cycle. Actin cytoskeleton has an integral role in establishing and spreading HIV-1 infection. Herein, we studied in vitro effects of E2 on HIV-1 infection and on CFL1 expression to gain insight into the mechanism of HIV-1 inhibition by E2. E2 dose-dependently inhibited HIV-1BaL infection in peripheral blood mononuclear cells (PBMCs) and endocervix. In PBMCs and endocervix, E2 increased protein expression of total CFL1 and phosphorylated CFL1 (pCFL1) and pCFL1/CFL1 ratios. LIMKi3, a LIM kinase 1 and 2 inhibitor, abrogated the phenotype and restored infection in both PBMCs and endocervix; inhibited E2-induced expression of total CFL1, pCFL1; and decreased pCFL1/CFL1 ratios. Knockdown of CFL1 in PBMCs also abrogated the phenotype and partially restored infection. Additional analysis of soluble mediators revealed decreased concentrations of pro-inflammatory chemokines CXCL10 and CCL5 in infected tissues incubated with E2. Our results suggest a link between E2-mediated anti-HIV-1 activity and expression of CFL1 in PBMCs and endocervical mucosa. The data support exploration of cytoskeletal signaling pathway targets for the development of prevention strategies against HIV-1.

The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis.

The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR.

Cellular heterogeneity and transcriptomic profiles during intrahepatic cholangiocarcinoma initiation and progression.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is not fully investigated, and how stromal cells contribute to ICC formation is poorly understood. We aimed to uncover ICC origin, cellular heterogeneity, and critical modulators during ICC initiation/progression, and to decipher how fibroblast and endothelial cells in the stromal compartment favor ICC progression. APPROACH AND RESULTS: We performed single-cell RNA sequencing (scRNA-seq) using AKT/Notch intracellular domain-induced mouse ICC tissues at early, middle, and late stages. We analyzed the transcriptomic landscape, cellular classification and evolution, and intercellular communication during ICC initiation/progression. We confirmed the findings using quantitative real-time PCR, western blotting, immunohistochemistry or immunofluorescence, and gene knockout/knockdown analysis. We identified stress-responding and proliferating subpopulations in late-stage mouse ICC tissues and validated them using human scRNA-seq data sets. By integrating weighted correlation network analysis and protein-protein interaction through least absolute shrinkage and selection operator regression, we identified zinc finger, MIZ-type containing 1 (Zmiz1) and Y box protein 1 (Ybx1) as core transcription factors required by stress-responding and proliferating ICC cells, respectively. Knockout of either one led to the blockade of ICC initiation/progression. Using two other ICC mouse models (YAP/AKT, KRAS/p19) and human ICC scRNA-seq data sets, we confirmed the orchestrating roles of Zmiz1 and Ybx1 in ICC occurrence and development. In addition, hes family bHLH transcription factor 1, cofilin 1, and inhibitor of DNA binding 1 were identified as driver genes for ICC. Moreover, periportal liver sinusoidal endothelial cells could differentiate into tip endothelial cells to promote ICC development, and this was Dll4-Notch4-Efnb2 signaling-dependent. CONCLUSIONS: Stress-responding and ICC proliferating subtypes were identified, and Zmiz1 and Ybx1 were revealed as core transcription factors in these subtypes. Fibroblast-endothelial cell interaction promotes ICC development.

Identification of the most damaging nsSNPs in the human CFL1 gene and their functional and structural impacts on cofilin-1 protein.

The cofilin-1 protein, encoded by CFL1, is an actin-binding protein that regulates F-actin depolymerization and nucleation activity through phosphorylation and dephosphorylation. CFL1 has been implicated in the development of neurodegenerative diseases (Alzheimer's disease and Huntington's disease), neuronal migration disorders (lissencephaly, epilepsy, and schizophrenia), and neural tube closure defects. Mutations in CFL1 have been associated with impaired neural crest cell migration and neural tube closure defects. In our study, various computational approaches were utilized to explore single-nucleotide polymorphisms (SNPs) in CFL1. The Variation Viewer and gnomAD databases were used to retrieve CFL1 SNPs, including 46 nonsynonymous SNPs (nsSNPs). The functional and structural annotation of SNPs was performed using 12 sequence-based web applications, which identified 20 nsSNPs as being the most likely to be deleterious or disease-causing. The conservation of cofilin-1 protein structures was illustrated using the ConSurf and PROSITE web servers, which projected the 12 most deleterious nsSNPs onto conserved domains, with the potential to disrupt the protein's functionality. These 12 nsSNPs were selected for protein structure construction, and the DynaMut/DUET servers predicted that the protein variants V7G, L84P, and L99A were the most likely to be damaging to the cofilin-1 protein structure or function. The evaluation of molecular docking studies demonstrated that the L99A and L84P cofilin-1 variants reduce the binding affinity for actin compared with the native cofilin-1 structure, and molecular dynamic simulation studies confirmed that these variants might destabilize the protein structure. The consequences of putative mutations on protein-protein interactions and post-translational modification sites in the cofilin-1 protein structure were analyzed. This study represents the first complete approach to understanding the effects of nsSNPs within the actin-depolymerizing factor/cofilin family, which suggested that SNPs resulting in L84P (rs199716082) and L99A (rs267603119) variants represent significant CFL1 mutations associated with disease development.

Identification and functional characterization of Cofilin-1 as a new member of antimicrobial protein.

Cofilin-1 (Cfl1), a member of the ADF/cofilin family, has been identified as one of differentially expressed proteins in human dendritic cells challenged with lipopolysaccharide (LPS), suggesting that it may be involved in immune response. Here we showed that zebrafish cfl1 was markedly up-regulated by LPS and LTA treatment. We also showed that zebrafish recombinant Cfl1 (rCfl1) not only bound to the Gram-negative and positive bacteria A. hydrophila and S. aureus as well as their signature molecules LPS and LTA but also inhibited the growth of the bacteria. Moreover, we found that the heparin-binding motif-containing regions of Cfl1, i.e., Cfl19-25, Cfl134-51 and Cfl1108-125, like rCfl1, were also able to bind to LPS and LTA and to inhibit the bacterial growth. rCfl1, Cfl19-25, Cfl134-51, and Cfl1108-125 were all able to cause bacterial cell destruction, to induce membrane depolarization, and to stimulate intracellular ROS production. Finally, we showed that zebrafish Cfl1 could protect developing embryos/larvae against attack by the potential pathogen A. hydrophila. These data together indicate that zebrafish Cfl1 plays an immune-relevant role as a newly-characterized antimicrobial protein.