Cytoskeleton Regulatory Protein Research Articles

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes.

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Effects of the oxoaporphine alkaloid hernandonine on dengue virus. Evidence for its mechanisms of action

BackgroundDengue, caused by the dengue virus (Orthoflavivirus dengue, encompassing DENV types 1-4), is a member of the Flaviviridae family. The symptoms of dengue range from subclinical or mild manifestations to potentially fatal complications. The management of severe dengue is exceptionally challenging due to the absence of effective antiviral medications. In this context, natural products, whether in the form of pure compounds or standardized plant extracts, have emerged as a promising source for the development of novel antiviral therapeutics. Hernandonine, an oxoaporphine alkaloid found in Hernandia nymphaeifolia (C. Presl) Kubitzki. serves both as a metabolite and an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase. PurposeThis study investigated the ability of hernandonine to inhibit DENV infection and explored its potential mechanisms. Study designTo assess the in vitro anti-DENV activity, cells or induced pluripotent stem cell (iPSC)-derived cerebral organoids were exposed to hernandonine before or after infection with DENV. Along with hernandonine, the endocytosis modulators, genistein, wortmannin, Methyl-β-cyclodextrin (MβCD) and lovastatin, were used in the assays. MethodsThe DENV infectivity and virion production in cells or cerebral organoids treated with compounds were determined. Various methods, including cell and cerebral organoids imaging, protein and gene detection were conducted to explore their antiviral mechanisms. ResultsThe results revealed notable antiviral properties of hernandonine, particularly in inhibiting DENV during the early stages of infection. Mechanistic analysis demonstrated that, akin to genistein, wortmannin, methyl-β-cyclodextrin (MβCD), and lovastatin, hernandonine exerted an influence on cholesterol-rich lipid rafts. It also restrained the pseudopodial movement ability of cells, potentially through the downregulation of cytoskeleton and endocytosis regulatory genes or protein expression. Moreover, hernandonine's virucidal activity was demonstrated. Hernandonine's inhibition of DENV infection was further validated in a disease-relevant iPSC-derived cerebral organoids model, a novel DENV-2 infection system worthy of further application. ConclusionThis study evidenced the potential of hernandonine as a novel candidate in the fight against DENV infection.

Construction and validation of a joint diagnosis model based on random forest and artificial intelligence network for hepatitis B-related hepatocellular carcinoma.

Hepatitis B virus (HBV) is the dominant pathogenic factor of hepatocellular carcinoma (HCC) in Asia and Africa. Early identification and clinical diagnosis are crucial for HBV-related HCC. Random forest (RF) and artificial neural network (ANN) were an innovative and highly effective supervised machine learning (ML) algorithm for the early diagnosis and screening of HBV-related HCC. This study aims to identify significant biomarkers and develop a novel genetic model for the efficient diagnosis of HBV-related HCC. Gene Expression Omnibus (GEO) Series (GSE)19665, GSE55092, and GSE121248 were used to identify significant differentially expressed genes (DEGs). The enrichment analysis was performed on Metascape online tool. The RF algorithm and ANN were used to select the potential predictive gene panels and construct an HBV-related HCC diagnostic model. Subsequently, GSE17548, GSE104310, GSE44074, and GSE136247 were used to test the accuracy of the ANN model. Finally, the CIBERSORT algorithm was used to assess the abundance of immune infiltrates in all samples. First, 116 genes were identified as DEGs, and the DEGs were particularly enriched in cellular hormone metabolic process, monocarboxylic acid metabolic process, NABA extracellular matrix (ECM) AFFILIATED steroid metabolic process and metabolism of bile acid and bile salt. DNA topoisomerase II alpha (TOP2A), C-type lectin domain family 1 member B (CLEC1B), BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B), ficolin 2 (FCN2), C-X-C motif chemokine ligand 14 (CXCL14), cyclase associated actin cytoskeleton regulatory protein 2 (CAP2), ficolin 3 (FCN3), kynurenine 3-monooxygenase (KMO) and cadherin related family member 2 (CDHR2) were available to develop an HBV-related HCC diagnostic model. After validation, the diagnostic model showed high sensitivity (88.5%, 90%, 88.5%, 76.5%) and specificity (100%, 81.8%, 89.5%, 72.2%), and the areas under the receiver operating characteristic (ROC) curves showed excellent efficiency (1, 0.927, 0.921, 0.833). Finally, the percentage of infiltrating immune cell types [B cells naïve, B cells memory, plasma cells, T cells CD8, T cells CD4 memory resting, T cells regulatory (Tregs), T cells gamma delta, natural killer (NK) cells resting, NK cells activated, Macrophages M0, Dendritic cells activated, Mast cells activated] for hepatitis B-related HCC were significantly different from that of non-cancerous liver tissue with HBV. A novel early diagnostic model of HBV-related HCC was established, and the model showed better efficiency in distinguishing HBV-related HCC from other non-cancerous with HBV individuals.

Characterization of the proteome changes in an in vitro hyperosmotic dry eye model employing human corneal epithelial cells

Aims/Purpose: The detrimental effects of tear hyperosmolarity on human corneal epithelial (HCE) cells is a primary cause of dry eye syndrome (DES). However, the molecular mechanisms underlying inflammatory processes and cellular dysfunction in HCE are largely unknown. This study characterized the proteome changes in an optimized hyperosmolarity‐induced DES model in vitro.Methods: An in vitro hyperosmolar (400–550 mOsm) DES model was established in a simian virus 40 (SV40)‐immortalized HCE cell line. Cells incubated in medium with physiological osmolarity (312 mOsm) were used as control. Cell viability, cell death and intracellular reactive oxygen species (ROS) levels were analysed with MTS, Annexin‐V‐FLUOS, crystal violet and DCFDA assays. Mass spectrometry‐based proteomics analyses characterized the proteome and elucidated the underlying molecular changes.Results: The HCE cell viability was significantly decreased in a concentration‐dependent manner with increasing hyperosmolarity compared to control at 48 hr (450 mOsm, −47%, p = 2.7 × 10−8; 500 mOsm, −71%, p = 1.1 × 10−4; 550 mOsm, −84%, p = 2.6 × 10−5). Notably, cell adherence was significantly decreased (p = 5.8 × 10−9), while levels of ROS (p = 3.6 × 10−2) and apoptosis (p = 3.6 × 10−2) were significantly increased at 450 mOsm compared to control. Proteomics analysis identified ~2000 proteins (FDR <1%) and, as many as 215 proteins were found to be significantly (p < 0.05) differentially abundant in the 450 vs. 312 mOsm. The effects of high hyperosmolarity (450 mOsm)‐induced damage resembling DES on the HCE cell were further demonstrated by the expression of proteins involved in the activation of inflammatory response (p = 5.5 × 10−3), apoptosis (p = 5.3 × 10−4) and generation of ROS (e.g., glutathione S‐transferase P) (p = 7.1 × 10−3). Moreover, many differentially expressed proteins were also implicated in the regulation of cell viability (p = 1.1 × 10−6) and organization of cytoplasm (e.g., cyclase associated actin cytoskeleton regulatory protein 1) (p = 2.4 × 10−2).Conclusions: In summary, an optimized hyperosmolarity‐induced DES cell culture model for proteomics analysis was established, and specific alterations in the proteome were characterized in this model. This in vitro model would be instrumental for testing therapeutic targets for DES in the future.

HMENA isoforms regulate cancer intrinsic type I IFN signaling and extrinsic mechanisms of resistance to immune checkpoint blockade in NSCLC

BackgroundUnderstanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the...

Cleavage of tropomodulin-3 by asparagine endopeptidase promotes cancer malignancy by actin remodeling and SND1/RhoA signaling

BackgroundAbnormal proliferation and migration of cells are hallmarks of cancer initiation and malignancy. Asparagine endopeptidase (AEP) has specific substrate cleavage ability and plays a pro-cancer role in a variety of cancers. However, the underlying mechanism of AEP in cancer proliferation and migration still remains unclear.MethodsCo-immunoprecipitation and following mass spectrometry were used to identify the substrate of AEP. Western blotting was applied to measure the expression of proteins. Single cell/nuclear-sequences were done to detect the heterogeneous expression of Tmod3 in tumor tissues. CCK-8 assay, flow cytometry assays, colony formation assay, Transwell assay and scratch wound-healing assay were performed as cellular functional experiments. Mouse intracranial xenograft tumors were studied in in vivo experiments.ResultsHere we showed that AEP cleaved a ubiquitous cytoskeleton regulatory protein, tropomodulin-3 (Tmod3) at asparagine 157 (N157) and produced two functional truncations (tTmod3-N and tTmod3-C). Truncated Tmod3 was detected in diverse tumors and was found to be associated with poor prognosis of high-grade glioma. Functional studies showed that tTmod3-N and tTmod3-C enhanced cancer cell migration and proliferation, respectively. Animal models further revealed the tumor-promoting effects of AEP truncated Tmod3 in vivo. Mechanistically, tTmod3-N was enriched in the cell cortex and competitively inhibited the pointed-end capping effect of wild-type Tmod3 on filamentous actin (F-actin), leading to actin remodeling. tTmod3-C translocated to the nucleus, where it interacted with Staphylococcal Nuclease And Tudor Domain Containing 1 (SND1), facilitating the transcription of Ras Homolog Family Member A/Cyclin Dependent Kinases (RhoA/CDKs).ConclusionThe newly identified AEP-Tmod3 protease signaling axis is a novel “dual-regulation” mechanism of tumor cell proliferation and migration. Our work provides new clues to the underlying mechanisms of cancer proliferation and invasive progression and evidence for targeting AEP or Tmod3 for therapy.

Whole Genome Re-sequencing and Bulk Segregant Analysis Reveals Chromosomal Location for Papaya Ringspot Virus W Resistance in Squash.

Squash (Cucurbita moschata) is among the most important cucurbit crops grown worldwide. Plant pathogen, Papaya ringspot virus W (PRSV-W) causes significant yield loss in commercial squash production globally. The development of virus-resistant cultivars can complement integrated disease management and mitigate losses due to viral infections. However, the genetic loci and molecular markers linked to PRSV-W resistance that could facilitate marker-assisted selection (MAS) for accelerated cultivar development are unknown. In this study, quantitative trait loci (QTL), molecular markers, and candidate genes associated with PRSV-W resistance in squash were identified in an F2 population (n = 118) derived from a cross between Nigerian Local accession (resistant) and Butterbush cultivar (susceptible). Whole genome re-sequencing-based bulked segregant analysis (QTLseq method; n = 10 for each bulk) and non-parametric interval mapping were used to identify a major QTL associated with PRSV-W resistance on chromosome 9 (QtlPRSV-C09) (p < 0.05) of C. moschata. QtlPRSV-C09 extended from 785,532 to 5,093,314 bp and harbored 12,245 SNPs among which 94 were high-effect variants. To validate QtlPRSV-C09, 13 SNP markers were assayed as Kompetitive allele-specific PCR (KASP) markers in the F2 population and tested for the association with PRSV-W resistance. Among these, two KASP markers (Ch09_2080834 and Ch09_5023865-1) showed significant association with PRSV-W resistance (p < 0.05). The two SNPs were located within exons of putative disease-resistant genes encoding the clathrin assembly family and actin cytoskeleton-regulatory complex proteins, which are implicated in disease resistance across plant species. The findings of this study will facilitate MAS for PRSV-W resistance in squash and allow further understanding of the functional mechanisms underlying potyvirus resistance in Cucurbita species.

A Comparative Study of the Role of Formins in Drosophila Embryonic Dorsal Closure.

Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal side of the Drosophila embryo. This process involves the coordination of several forces generated in the epidermal cell layer and in the amnioserosa cells, covering the hole. Ultimately, these forces arise due to cytoskeletal rearrangements that induce changes in cell shape and result in tissue movement. While a number of cytoskeleton regulatory proteins have already been linked to dorsal closure, here we expand this list by demonstrating that four of the six Drosophila formin type actin assembly factors are needed to bring about the proper fusion of the epithelia. An analysis of the morphological and dynamic properties of dorsal closure in formin mutants revealed a differential contribution for each formin, although we found evidence for functional redundancies as well. Therefore, we propose that the four formins promote the formation of several, and only partly identical, actin structures each with a specific role in the mechanics of dorsal closure.

CORO1A regulates lipoprotein uptake in Leydig cells exposed to cadmium

Cadmium (Cd) is one of the most common environmental pollutants, which has a long biological half-life. Maternal Cd-exposure in the natural environment causes steroidogenesis defects resulting in spermatogenesis disorder in male offspring. For better understanding its underlying mechanism, we have employed iTRAQ to screen the differentially expressed protein and found that the expression of CORO1A and Cofilin 1 was up-regulated approximately 2 fold in Leydig cells of maternal Cd-exposure offspring. As the major source of steroid hormone, cholesterol is transported to cells via receptor-mediated endocytosis which relies on the remodel of cytoskeleton, then stores in lipid droplets (LDs). However, few studies have focused on the role of cytoskeleton in abnormal steroidogenesis. This study was performed to explore the role of CORO1A in androgen deficiency caused by Cd exposure and its involvement of low-density lipoprotein (LDL) uptake and effects on LDs. We found that Cd resulted in the up-regulation of CORO1A and Cofilin 1, and down-regulation of Profilin 1 in the testis of male offspring with maternal exposure. The structure of filamentous actin was broken, disordered and even crumpled up in Cd-treated R2C cells. F-actin disassembly led to a low uptake of LDL with a reduced number of LDs, followed by decreased total cholesterol and low progesterone production. When CORO1A was silenced, the expression of Cofilin 1 was down-regulated and Profilin 1 was up-regulated in Cd-treated R2C cells. The filamentous actin was rescued and the integrated cytoskeleton prompted LDL uptake, which resulted in the increased total cholesterol and high progesterone production. These findings highlight the crucial role of CORO1A as a cytoskeleton regulatory protein in steroidogenesis, which may help to better understand Cd-induced steroid hormone deficiency in children.

Deletion of Calponin 2 Reduces the Formation of Postoperative Peritoneal Adhesions

Aim of the study: Postoperative peritoneal adhesions are a common cause of morbidity after surgery, resulting in multiple complications. Macrophage-mediated inflammation and myofibroblast differentiation after tissue injury play central roles in the pathogenesis and progression of adhesion formation. Calponin 2 is an actin cytoskeleton regulatory protein in endothelial cells, macrophages and fibroblasts that are key players in the development of fibrosis. Deletion of calponin 2 has been shown to attenuate inflammatory arthritis, atherosclerosis and fibrocalcification of the aortic valves. The present study investigated the effect of calponin 2 deletion on attenuating the formation of peritoneal adhesions in a mouse model for potential use as a new therapeutic target. Materials and methods: Sterile surgical procedures under general anesthesia were used on paired wild type (WT) and calponin 2 knockout (KO) mice to generate mild injury on the cecal and abdominal wall peritonea. Three and seven days post-operation, the mice were compared postmortem for the formation of peritoneal adhesions. Tissues at the adhesion sites were examined with histology and immunofluorescent studies for macrophage and myofibroblast activations. Results: Quantitative scoring demonstrated that calponin 2 KO mice developed significantly less postoperative peritoneal adhesions than that in WT mice. Calponin 2 deletion resulted in less infiltration of F4/80+ macrophages at the adhesion sites with less myofibroblast differentiation and collagen deposition than WT controls. Conclusions: The data show that deletion of calponin 2 effectively reduces postoperative peritoneal adhesion, presenting a novel molecular target for clinical prevention.

Microtubules, actin and cytolinkers: how to connect cytoskeletons in the neuronal growth cone

Cytolinkers ensure the integration of the different cytoskeleton components in the neuronal growth cone during development and in the course of axon regeneration. In neurons, an integrated skeleton guarantees appropriate function, and connectivity of high order circuits. Over the past years, several cytoskeleton regulatory proteins with actin-microtubule crosslinking activity have been identified. In neurons, the importance of spectrins, formins and other cytolinkers capable of coupling actin and microtubules has been extensively highlighted during axon outgrowth and guidance. In this Review, we discuss the current knowledge on cytolinkers specifically expressed in the neuronal growth cone of developing and regenerating axons.

Mechanism of WASP and WAVE family proteins in the progression of prostate cancer.

Prostate cancer (PCa) is the second most commonly diagnosed and third lethal cause of death from cancer in men worldwide. Despite the availability of vast treatment procedures, still the high occurrence of invasion and metastasis of PCa are reported in cancer patients. The WASP (Wiskott-Aldrich syndrome protein) and WAVE (WASP family verprolin homologous protein) family of proteins are actin cytoskeleton regulatory proteins, reported to enhance cancer cell invasion and migration in prostate cancer. Hence, this review sheds light on the studies that explored the potential role of WASP and WAVE family of proteins in invasion and metastasis of prostate cancer. The research articles explored for the completion of this review were mostly from PubMed and Google Scholar by using the appropriate keywords for indexing. The conserved function of WASP and WAVE protein family is to receive the upstream signals from the Rho GTPase family and transmit them to activate the Arp2/3 complex that leads to rapid actin polymerization at leading edge of cells, which is crucial for PCa metastasis. Therefore, targeting these proteins could reflect a very interesting therapeutic opportunity to combat prostate cancer.

Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells

Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel-Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1-actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC.

Upregulation of cyclase-associated actin cytoskeleton regulatory protein 2 in epithelial ovarian cancer correlates with aggressive histologic types and worse outcomes.

Cyclase-associated actin cytoskeleton regulatory protein 2 (CAP2) regulates actin dynamics to control cell cycles and cell migration. CAP2 overexpression contributes to cancer progression in several tumor types; however, the role of CAP2 expression in ovarian cancer remains unclear. This study aimed to clarify the significance of CAP2 expression in epithelial ovarian tumor. We evaluated CAP2 expression in ovarian cancer cell lines using quantitative real-time polymerase chain reaction, western blotting and immunocytochemistry and examined the effect of CAP2 silencing in migration and proliferation assays. CAP2 immunohistochemistry was conducted using tissue specimens from 432 ovarian carcinoma patients; a further 55 borderline and benign 65 lesions were analyzed. CAP2 expression levels were defined as low, intermediate or high, for correlation analysis with clinicopathological factors. CAP2 expression was significantly higher in cell lines from Type II ovarian cancer than in those in Type I, and knockdown of CAP2 showed decreased migration and proliferation. Higher levels of CAP2 expression in human tissues were associated with Type II histology, residual lesion, lymph node metastasis, ascites cytology and higher clinical stage. High CAP2 expression levels were observed in 26 (23.4%) of 111 Type II ovarian cancers and in 16 (5.0%) of 321 Type I cancers but not in any borderline or benign lesions. Multivariate analyses showed that CAP2 expression in ovarian cancer is an independent prognostic factor for recurrence-free survival (P=0.019). CAP2 expression is upregulated in aggressive histologic types of epithelial ovarian cancer and serves as a novel prognostic biomarker for patient survival.

PLA2G4A Is a Potential Biomarker Predicting Shorter Overall Survival in Patients with Non-M3/NPM1 Wildtype Acute Myeloid Leukemia.

In this study, we aimed at exploring and validating the prognostic value of PLA2G4A expression in patients with non-M3/nucleophosmin (NPM1) wildtype (WT) acute myeloid leukemia (AML) by using two independent datasets. Data from the Cancer Genome Atlas-acute myeloid leukemia (TCGA-LAML) and the therapeutically applicable research to generate effective treatments (TARGET)-AML were used to assess the prognostic value of PLA2G4A in NPM1-WT AML cases. Results showed that non-M3 AML cases had significantly increased PLA2G4A expression compared with normal peripheral blood samples. Patients with high PLA2G4A expression (separated by median gene expression) had a significantly shorter overall survival (OS) compared with the group with low PLA2G4A expression, in both TCGA-LAML and TARGET-AML. Multivariate analysis showed that high PLA2G4A expression was independently associated with shorter OS in 97 non-M3/NPM1-WT AML cases in TCGA-LAML (hazard ratio [HR]: 1.946, 95% confidence interval [CI]: 1.094-3.462, q = 0.036). The prognostic value was validated based on 120 primary non-M3/NPM1-WT AML cases in TARGET-AML (HR: 1.518, 95% CI: 1.037-2.223, q = 0.048). Therefore, PLA2G4A expression might serve as an independent prognostic marker in OS in patients with non-M3/NPM1 WT AML. Bioinformatic analysis identified that several proteins physically interacted with PLA2G4A, some of which have well-characterized oncogenic properties in AML, such as RUVBL2, cytoskeleton regulatory protein 1 (CAP1), signal transducer and activator of transcription 3 (STAT3), and MYCBP. Therefore, we hypothesized that PLA2G4A upregulation has multiple effects on the malignant phenotype of AML cells together with its partners. Future molecular studies are required to explore the detailed regulatory network involved.

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

ABSTRACT T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule-organizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1 phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1 phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1 re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1 regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization. Abbreviations Ab, antibody; AICD, activation-induced cell death; AIP, average intensity projection; APC, antigen-presenting cell; BCR, B-cell receptor for antigen; C, centre of mass; cent2, centrin 2; cIS, central region of the immune synapse; CMAC, CellTracker™ Blue (7-amino-4-chloromethylcoumarin); cSMAC, central supramolecular activation cluster; CTL, cytotoxic T lymphocytes; DAG, diacylglycerol; DGKα, diacylglycerol kinase α; Dia1, Diaphanous-1; dSMAC, distal supramolecular activation cluster; ECL, enhanced chemiluminescence; ESCRT, endosomal sorting complex required for traffic; F-actin, filamentous actin; Fact-low cIS, F-actin-low region at the centre of the immune synapse; FasL, Fas ligand; FMNL1, formin-like 1; fps, frames per second; GFP, green fluorescent protein; HBSS, Hank’s balanced salt solution; HRP, horseradish peroxidase; ILV, intraluminal vesicles; IS, immune synapse; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MIP, maximal intensity projection; MVB, multivesicular bodies; MTOC, microtubule-organizing centre; NS, not significant; PBL, peripheral blood lymphocytes; PKC, protein kinase C; PKCδ, protein kinase C δ isoform; PLC, phospholipase C; PMA, phorbol myristate acetate; Pol. Index, polarization index; pSMAC, peripheral supramolecular activation cluster; PSF, point spread function; ROI, region of interest; SD, standard deviation; shRNA, short hairpin RNA; SEE, Staphylococcus enterotoxin E; SMAC, supramolecular activation cluster; TCR, T-cell receptor for antigen; T-helper (Th); TRANS, transmittance; WB, Western blot.

MiR-409-5p as a Regulator of Neurite Growth Is Down Regulated in APP/PS1 Murine Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a heterogeneous neurodegenerative disease. Recent studies suggest that miRNA expression changes are associated with the development of AD. Our previous study showed that the expression level of miR-409-5p was stably downregulated in the early stage of APP/PS1 double transgenic mice model of AD. We now report that miR-409-5p impairs neurite outgrowth, decreases neuronal viability, and accelerates the progression of Aβ1–42-induced pathologies. In this study, we found that Aβ1–42 peptide significantly decreased the expression of miR-409-5p, which was consistent with the expression profile of miR-409-5p in the APP/PS1 mice cortexes. Plek was confirmed to be a potential regulatory target of miR-409-5p by luciferase assay and Western blotting. Overexpression of miR-409-5p has an obvious neurotoxicity in neuronal cell viability and differentiation, whereas Plek overexpression could partially rescue neurite outgrowth from this toxicity. Some cytoskeleton regulatory proteins have been found to be related to AD pathogenesis. Our data show some clues that cytoskeletal reorganization may play roles in AD pathology. The early downregulation of miR-409-5p in AD progression might be a self-protective reaction to alleviate the synaptic damage induced by Aβ, which may be used as a potential early biomarker of AD.

Hydrogen treatment prevents lipopolysaccharide-induced pulmonary endothelial cell dysfunction through RhoA inhibition

BackgroundPulmonary microvascular endothelial cells (PMVECs) are initial targets of sepsis-induced acute lung injury (ALI). During the apoptosis of PMVECs, tight junctions (TJ) and adherens junctions (AJ) are firstly damaged. Previous studies have suggested hydrogen treatment can protect lung microvasculature of mice from sepsis-induced endothelial dysfunction and maintain the coherence of pulmonary endothelium, but the underlying mechanism remains unclear. MethodsWe investigated the role of hydrogen-rich medium on regulating intercellular junction proteins under lipopolysaccharide (LPS) treatment which mimicked sepsis in vitro. Changes of cytoskeleton regulatory protein ROCK and RhoA as well as PMVEC apoptotic rate were examined. ResultsLPS treatment reduced the expression levels of occludin and VE-cadherin in PMVECs, while hydrogen-rich medium can recover these changes. Furthermore, H2 can significantly ameliorate the excessive expression of ROCK and RhoA under sepsis-mimicking condition. The application of RhoA activator U-46619 resulted in a more significant elevation in cell apoptotic rate as well as reduction in the expression of junctional proteins. Using H2 can almost completely inhibit the effects of RhoA activator. ConclusionsOur findings suggest that RhoA is a crucial protein in the signaling pathway of LPS-induced endothelial cell dysfunction. Hydrogen treatment can prevent LPS-induced junctional injury and cell death by inhibiting the activity of RhoA.

Impaired vascular smooth muscle cell force-generating capacity and phenotypic deregulation in Marfan Syndrome mice

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.