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1.
Mol Ther ; 28(4): 1092-1104, 2020 04 08.
Article in English | MEDLINE | ID: mdl-32087767

ABSTRACT

The N-degron pathway is an emerging target for anti-tumor therapies, because of its capacity to positively regulate many hallmarks of cancer, including angiogenesis, cell proliferation, motility, and survival. Thus, inhibition of the N-degron pathway offers the potential to be a highly effective anti-cancer treatment. With the use of a small interfering RNA (siRNA)-mediated approach for selective downregulation of the four Arg/N-degron-dependent ubiquitin ligases, UBR1, UBR2, UBR4, and UBR5, we demonstrated decreased cell migration and proliferation and increased spontaneous apoptosis in cancer cells. Chronic treatment with lipid nanoparticles (LNPs) loaded with siRNA in mice efficiently downregulates the expression of UBR-ubiquitin ligases in the liver without any significant toxic effects but engages the immune system and causes inflammation. However, when used in a lower dose, in combination with a chemotherapeutic drug, downregulation of the Arg/N-degron pathway E3 ligases successfully reduced tumor load by decreasing proliferation and increasing apoptosis in a mouse model of hepatocellular carcinoma, while avoiding the inflammatory response. Our study demonstrates that UBR-ubiquitin ligases of the Arg/N-degron pathway are promising targets for the development of improved therapies for many cancer types.


Subject(s)
Carcinoma, Hepatocellular/drug therapy , Down-Regulation , Doxorubicin/administration & dosage , Liver Neoplasms/drug therapy , RNA, Small Interfering/administration & dosage , Ubiquitin-Protein Ligases/genetics , Animals , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Doxorubicin/pharmacology , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Liposomes , Liver Neoplasms/genetics , Mice , Nanoparticles , RNA, Small Interfering/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Xenograft Model Antitumor Assays
2.
Int J Mol Sci ; 19(7)2018 06 21.
Article in English | MEDLINE | ID: mdl-29933613

ABSTRACT

Vascular endothelial growth factor (VEGF) is important for maintaining healthy endothelium, which is crucial for vascular integrity. In this paper, we show that VEGF stimulates the nuclear translocation of endothelial differentiation-related factor 1 (EDF1), a highly conserved intracellular protein implicated in molecular events that are pivotal to endothelial function. In the nucleus, EDF1 serves as a transcriptional coactivator of peroxisome proliferator-activated receptor gamma (PPARγ), which has a protective role in the vasculature. Indeed, silencing EDF1 prevents VEGF induction of PPARγ activity as detected by gene reporter assay. Accordingly, silencing EDF1 markedly inhibits the stimulatory effect of VEGF on the expression of FABP4, a PPARγ-inducible gene. As nitric oxide is a marker of endothelial function, it is noteworthy that we report a link between EDF1 silencing, decreased levels of FABP4, and nitric oxide production. We conclude that EDF1 is required for VEGF-induced activation of the transcriptional activity of PPARγ.


Subject(s)
Calmodulin-Binding Proteins/genetics , Fatty Acid-Binding Proteins/genetics , Human Umbilical Vein Endothelial Cells/drug effects , PPAR gamma/genetics , Vascular Endothelial Growth Factor A/pharmacology , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Fatty Acid-Binding Proteins/metabolism , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Nitric Oxide/biosynthesis , PPAR gamma/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Transcription, Genetic , Vascular Endothelial Growth Factor A/metabolism
3.
Elife ; 72018 05 14.
Article in English | MEDLINE | ID: mdl-29757140

ABSTRACT

The vascular pathogen Verticillium dahliae infects the roots of plants to cause Verticillium wilt. The molecular mechanisms underlying V. dahliae virulence and host resistance remain elusive. Here, we demonstrate that a secretory protein, VdSCP41, functions as an intracellular effector that promotes V. dahliae virulence. The Arabidopsis master immune regulators CBP60g and SARD1 and cotton GhCBP60b are targeted by VdSCP41. VdSCP41 binds the C-terminal portion of CBP60g to inhibit its transcription factor activity. Further analyses reveal a transcription activation domain within CBP60g that is required for VdSCP41 targeting. Mutations in both CBP60g and SARD1 compromise Arabidopsis resistance against V. dahliae and partially impair VdSCP41-mediated virulence. Moreover, virus-induced silencing of GhCBP60b compromises cotton resistance to V. dahliae. This work uncovers a virulence strategy in which the V. dahliae secretory protein VdSCP41 directly targets plant transcription factors to inhibit immunity, and reveals CBP60g, SARD1 and GhCBP60b as crucial components governing V. dahliae resistance.


Subject(s)
Arabidopsis Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/antagonists & inhibitors , Fungal Proteins/metabolism , Host-Pathogen Interactions , Plant Diseases/microbiology , Plant Immunity , Verticillium/pathogenicity , Arabidopsis , Arabidopsis Proteins/genetics , Calmodulin-Binding Proteins/genetics , DNA Mutational Analysis , Gossypium , Immune Evasion , Protein Binding , Verticillium/growth & development , Virulence
4.
J Mol Biol ; 430(6): 793-805, 2018 03 16.
Article in English | MEDLINE | ID: mdl-29408381

ABSTRACT

Titin-like kinases are muscle-specific kinases that regulate mechanical sensing in the sarcomere. Twitchin kinase (TwcK) is the best-characterized member of this family, both structurally and enzymatically. TwcK activity is auto-inhibited by a dual intrasteric mechanism, in which N- and C-terminal tail extensions wrap around the kinase domain, blocking the hinge region, the ATP binding pocket and the peptide substrate binding groove. Physiologically, kinase activation is thought to occur by a stretch-induced displacement of the inhibitory tails from the kinase domain. Here, we now show that TwcK inhibits its catalysis even in the absence of regulatory tails, by undergoing auto-phosphorylation at mechanistically important elements of the kinase fold. Using mass spectrometry, site-directed mutagenesis and catalytic assays on recombinant samples, we identify residues T212, T301, T316 and T401 as primary auto-phosphorylation sites in TwcK in vitro. Taken together, our results suggest that residue T316, located in the peptide substrate binding P+1 loop, is the dominantly regulatory site in TwcK. Based on these findings, we conclude that TwcK is regulated through a triple-inhibitory mechanism consisting of phosphorylation and intrasteric blockage, which is responsive not only to mechanical cues but also to biochemical modulation. This implies that mechanically stretched conformations of TwcK do not necessarily correspond to catalytically active states, as previously postulated. This further suggests a phosphorylation-dependent desensitization of the TwcK-mediated mechanoresponse of the sarcomere in vivo.


Subject(s)
Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/metabolism , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Muscle Proteins/antagonists & inhibitors , Muscle Proteins/metabolism , Amino Acid Sequence , Caenorhabditis elegans Proteins/genetics , Calmodulin-Binding Proteins/genetics , Carrier Proteins , Catalysis , Crystallography, X-Ray , Models, Molecular , Muscle Proteins/genetics , Mutagenesis, Site-Directed , Phosphorylation , Protein Conformation , Protein Domains , Protein Isoforms , Recombinant Proteins
5.
Int J Med Sci ; 15(1): 69-76, 2018.
Article in English | MEDLINE | ID: mdl-29333089

ABSTRACT

Objective: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high rate of mortality. Our previous study shows the expression of calponin 2 (CNN2) is up-regulated in hepatocellular carcinoma tissues, especially in metastatic ones. To better understand the role of CNN2 in HCC, RNA interference (RNAi) was used to explore its role in tumor growth and metastasis. Methods: Lentivirus-mediated CNN2-shRNA was transfected into SK-hep-1 cells, and the efficacy of CNN2 expression, cell migration, invasion, proliferation and cell cycles were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), Transwell assay, methyl thiazol tetrazolium assay and flow cytometry, respectively. SK-hep-1 cells transfected with Lentivirus-CNN2 shRNA were xenografted in Balb/C nude mice to explore the effect of CNN2-shRNA in tumor growth. Xenograft tumor tissues were examined for their histopathology, cell apoptosis, the expression of total protein and their corresponding phosphorylated protein of MEK1/2, ERK1/2, AKT, by hematoxylin and eosin stain (H & E staining), TUNEL assay, immunohistochemical technique, respectively. Results: Our research shows it is evident that CNN2 shRNA can effectively down-regulate the expressions of CNN2 mRNA and protein, inhibit cell proliferations, arrest cell cycles at the S phase and reduce cell migration and invasion. SK-hep-1 cells with CNN2 down-regulation have markedly attenuated tumor growth in nude mice. Xenograft tumor tissues have displayed typical tumor characteristics and no apoptosis is detected in shRNA group or in control group. No metastatic tumor was found in any group of nude mice. With CNN2 protein down-regulation, the protein of pMEK1/2 and pERK1/2 are effectively down-regulated, except pAKT, AKT, MEK1/2 and ERK1/2. Conclusions: CNN2 plays an important role in tumor growth and metastasis, possibly through MEK1/2-ERK1/2 signaling pathway. Our study illustrate that CNN2 might be a potential target in HCC molecular target therapy.


Subject(s)
Calcium-Binding Proteins/metabolism , Calmodulin-Binding Proteins/metabolism , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Microfilament Proteins/metabolism , Animals , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/genetics , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Down-Regulation , Female , Gene Knockdown Techniques , Genetic Vectors/genetics , Humans , Lentivirus/genetics , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , MAP Kinase Signaling System/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/genetics , Molecular Targeted Therapy/methods , Phosphorylation , RNA Interference , RNA, Small Interfering/genetics , S Phase Cell Cycle Checkpoints/genetics , Transfection/methods , Xenograft Model Antitumor Assays , Calponins
6.
Cell Rep ; 14(3): 598-610, 2016 Jan 26.
Article in English | MEDLINE | ID: mdl-26776507

ABSTRACT

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.


Subject(s)
Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS/metabolism , Base Sequence , Binding Sites , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Cell Line, Tumor , Cell Survival , Exons , Gene Expression Regulation, Neoplastic , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism , Humans , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/genetics , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/metabolism , Proto-Oncogene Protein c-fli-1/antagonists & inhibitors , Proto-Oncogene Protein c-fli-1/genetics , RNA Interference , RNA Precursors/metabolism , RNA Splicing , RNA Splicing Factors , RNA, Small Interfering/metabolism , RNA-Binding Protein EWS/antagonists & inhibitors , RNA-Binding Protein EWS/genetics , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Ribonucleoprotein, U2 Small Nuclear/antagonists & inhibitors , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism , Sarcoma, Ewing/pathology , Trans-Activators , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism
7.
Oncogene ; 34(11): 1432-41, 2015 Mar 12.
Article in English | MEDLINE | ID: mdl-24704824

ABSTRACT

Aberrant splicing of the cyclin-dependent kinase-associated phosphatase, KAP, promotes glioblastoma invasion in a Cdc2-dependent manner. However, the mechanism by which this occurs is unknown. Here we show that miR-26a, which is often amplified in glioblastoma, promotes invasion in phosphatase and tensin homolog (PTEN)-competent and PTEN-deficient glioblastoma cells by directly downregulating KAP expression. Mechanistically, we find that KAP binds and activates ROCK2. Thus, RNA-mediated downregulation of KAP leads to decreased ROCK2 activity and this, in turn, increases Rac1-mediated invasion. In addition, the decrease in KAP expression activates the cyclin-dependent kinase, Cdk2, and this directly promotes invasion by increasing retinoblastoma phosphorylation, E2F-dependent Cdc2 expression and Cdc2-mediated inactivation of the actomyosin inhibitor, caldesmon. Importantly, glioblastoma cell invasion mediated by this pathway can be antagonized by Cdk2/Cdc2 inhibitors in vitro and in vivo. Thus, two distinct RNA-based mechanisms activate this novel KAP/ROCK2/Cdk2-dependent invasion pathway in glioblastoma.


Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , Dual-Specificity Phosphatases/metabolism , Glioblastoma/pathology , MicroRNAs/physiology , rho-Associated Kinases/metabolism , Actomyosin/antagonists & inhibitors , Brain Neoplasms/pathology , CDC2 Protein Kinase , Calmodulin-Binding Proteins/antagonists & inhibitors , Cell Line, Tumor , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase Inhibitor Proteins/biosynthesis , Cyclin-Dependent Kinase Inhibitor Proteins/genetics , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/biosynthesis , Dual-Specificity Phosphatases/biosynthesis , Dual-Specificity Phosphatases/genetics , E2F Transcription Factors/physiology , Enzyme Activation , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Neoplasm Invasiveness , PTEN Phosphohydrolase/metabolism , Phosphorylation , Protein Binding , RNA Interference , RNA, Small Interfering , Retinoblastoma Protein/metabolism , rac1 GTP-Binding Protein/physiology
8.
Proc Natl Acad Sci U S A ; 110(10): 3800-5, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23431188

ABSTRACT

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.


Subject(s)
Microtubule-Associated Proteins/physiology , Ubiquitin-Protein Ligases/physiology , Yolk Sac/blood supply , Yolk Sac/enzymology , Animals , Autophagy/genetics , Autophagy/physiology , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cytoskeletal Proteins/antagonists & inhibitors , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/physiology , Embryonic Development/genetics , Embryonic Development/physiology , Endoderm/blood supply , Endoderm/cytology , Endoderm/enzymology , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , Mesoderm/blood supply , Mesoderm/cytology , Mesoderm/enzymology , Metabolic Networks and Pathways , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/genetics , Neovascularization, Physiologic/genetics , Pregnancy , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Yolk Sac/cytology , Yolk Sac/embryology
9.
J Proteome Res ; 12(2): 980-90, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23265641

ABSTRACT

In this study, we aim to identify biomarkers for gastric cancer metastasis using a quantitative proteomics approach. The proteins extracted from a panel of 4 gastric cancer cell lines, two derived from primary cancer (AGS, FU97) and two from lymph node metastasis (AZ521, MKN7), were labeled with iTRAQ (8-plex) reagents and analyzed by 2D-LC-MALDI-TOF/TOF MS. In total, 641 proteins were identified with at least a 95% confidence. Using cutoff values of >1.5 and <0.67, 19 proteins were found to be up-regulated and 34 were down-regulated in the metastatic versus primary gastric cancer cell lines respectively. Several of these dysregulated proteins, including caldesmon, were verified using Western blotting. It was found that caldesmon expression was decreased in the two metastasis-derived cell lines, and this was confirmed by further analysis of 7 gastric cancer cell lines. Furthermore, immunohistochemical staining of 9 pairs of primary gastric cancer and the matched lymph node metastasis tissue also corroborated this observation. Finally, knockdown of caldesmon using siRNA in AGS and FU97 gastric cancer cells resulted in an increase in cell migration and invasion, while the overexpression of caldesmon in AZ521 cells led to a decrease in cell migration and invasion. This study has thus established the potential role of caldesmon in gastric cancer metastasis, and further functional studies are underway to delineate the underlying mechanism of action of this protein.


Subject(s)
Biomarkers, Tumor/genetics , Calmodulin-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Lymphatic Metastasis/genetics , Neoplasm Proteins/genetics , Stomach Neoplasms/genetics , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/metabolism , Blotting, Western , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Cell Line, Tumor , Cell Movement , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Immunohistochemistry , Lymphatic Metastasis/diagnosis , Neoplasm Proteins/metabolism , RNA, Small Interfering/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Stomach Neoplasms/diagnosis , Stomach Neoplasms/metabolism
10.
J Neurosci ; 32(42): 14583-91, 2012 Oct 17.
Article in English | MEDLINE | ID: mdl-23077044

ABSTRACT

Glucocorticoids (GCs) mediate the effects of stress to cause structural plasticity in brain regions such as the hippocampus, including simplification of dendrites and shrinkage of dendritic spines. However, the molecular mechanics linking stress and GCs to these effects remain largely unclear. Here, we demonstrated that corticosterone (CORT) reduces the expression levels of caldesmon (CaD), causing dendritic spines to become vulnerable. CaD regulates cell motility by modulating the actin-myosin system and actin filament stability. In cultured rat hippocampal neurons, CaD localized to dendritic spines by binding to filamentous actin (F-actin), and CaD expression levels increased during spine development. CaD stabilized the F-actin dynamics in spines, thereby enlarging the spine heads, whereas CaD knockdown decreased the spine-head size via destabilization of the F-actin dynamics. CaD was also required for chemical LTP-induced actin stabilization. The CaD expression levels were markedly decreased by exposure to CORT mediated by suppression of serum response factor-dependent transcription. High CORT levels reduced both the spine-head size and F-actin stability similarly to CaD knockdown, and overexpressing CaD abolished the detrimental effect of CORT on dendritic spine development. These results indicate that CaD enlarges the spine-head size by stabilizing F-actin dynamics, and that CaD is a critical target in the GC-induced detrimental effects on dendritic spine development.


Subject(s)
Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/biosynthesis , Corticosterone/pharmacology , Dendritic Spines/physiology , Down-Regulation/genetics , Gene Expression Regulation, Developmental/physiology , Neurogenesis/genetics , Actins/antagonists & inhibitors , Actins/metabolism , Animals , Calmodulin-Binding Proteins/genetics , Cells, Cultured , Dendritic Spines/drug effects , Down-Regulation/drug effects , Humans , Male , Neurogenesis/drug effects , Protein Binding/drug effects , Protein Binding/genetics , Random Allocation , Rats , Rats, Wistar
11.
Microb Pathog ; 52(3): 149-56, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22197999

ABSTRACT

Recent work has demonstrated that the spectrin cytoskeleton is a host cell target, exploited during intestinal bacterial disease. Here we show that the highly virulent intestinal pathogen enterohaemorrhagic Escherichia coli (EHEC) is also reliant upon the spectrin cytoskeleton during key pathogenic events. Immunofluorescent microscopy demonstrated that the core components of the spectrin cytoskeleton (spectrin, adducin, and protein 4.1 [p4.1]) are recruited to sites of EHEC attachment and localized at pedestal structures along with the EHEC pedestal specific proteins IRSp53 and IRTKS. Further studies involving siRNA-mediated knockdowns of spectrin, adducin, or p4.1 revealed that those proteins are needed for efficient docking of EHEC to host cells, are involved in recruiting IRSp53 to the pedestal and are necessary for pedestal formation. These findings identify the spectrin cytoskeleton as a major host cell cytoskeletal network involved in critical EHEC pathogenic events.


Subject(s)
Bacterial Adhesion , Calmodulin-Binding Proteins/metabolism , Enterohemorrhagic Escherichia coli/pathogenicity , Host-Pathogen Interactions , Membrane Proteins/metabolism , Spectrin/metabolism , Calmodulin-Binding Proteins/antagonists & inhibitors , Gene Silencing , HeLa Cells , Humans , Membrane Proteins/antagonists & inhibitors , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Spectrin/antagonists & inhibitors
12.
Mol Cell Biol ; 30(21): 4980-95, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20733006

ABSTRACT

We have recently shown that Src induces the formation of podosomes and cell invasion by suppressing endogenous p53, while enhanced p53 strongly represses the Src-induced invasive phenotype. However, the mechanism by which Src and p53 play antagonistic roles in cell invasion is unknown. Here we show that the Stat3 oncogene is a required downstream effector of Src in inducing podosome structures and related invasive phenotypes. Stat3 promotes Src phenotypes through the suppression of p53 and the p53-inducible protein caldesmon, a known podosome antagonist. In contrast, enhanced p53 attenuates Stat3 function and Src-induced podosome formation by upregulating the tumor suppressor PTEN. PTEN, through the inactivation of Src/Stat3 function, also stabilizes the podosome-antagonizing p53/caldesmon axis, thereby further enhancing the anti-invasive potential of the cell. Furthermore, the protein phosphatase activity of PTEN plays a major role in the negative regulation of the Src/Stat3 pathway and represses podosome formation. Our data suggest that cellular invasiveness is dependent on the balance between two opposing forces: the proinvasive oncogenes Src-Stat3 and the anti-invasive tumor suppressors p53-PTEN.


Subject(s)
Cell Movement/physiology , PTEN Phosphohydrolase/physiology , STAT3 Transcription Factor/physiology , Tumor Suppressor Protein p53/physiology , src-Family Kinases/physiology , 3T3 Cells , Animals , Base Sequence , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/physiology , Cell Line , Cell Movement/genetics , DNA Primers/genetics , Gene Knockdown Techniques , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/physiology , Matrix Metalloproteinase 10/genetics , Matrix Metalloproteinase 10/physiology , Matrix Metalloproteinase Inhibitors , Mice , Models, Biological , Mutant Proteins/genetics , Mutant Proteins/physiology , Myocytes, Smooth Muscle/physiology , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/physiopathology , PTEN Phosphohydrolase/genetics , Phenotype , RNA, Small Interfering/genetics , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , Signal Transduction , Tumor Suppressor Protein p53/genetics , src-Family Kinases/genetics
13.
Biochemistry ; 49(19): 4191-9, 2010 May 18.
Article in English | MEDLINE | ID: mdl-20402494

ABSTRACT

Major contractile proteins were purified from relaxed actomyosin extracted from molluscan catch muscle myofibrils using ammonium sulfate fractionation and divalent cation precipitation. A fraction of this actomyosin was precipitated and purified as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). Another TW-MR complex was obtained via the removal of myosin. These supramolecular complexes and filaments assembled from purified myosin contained an endogenous protein kinase that phosphorylated myosin and myorod. Significantly, the activity of this novel myosin-associated (MA) kinase was inhibited at calcium concentrations of >0.1 microM. After partial purification of the kinase, we established that the inhibition resulted from binding of calcium to the substrate (myosin) and not from the binding to the enzyme (kinase). No inhibition was observed when myorod was used as a substrate, although the latter is identical to the rod portion of myosin lacking the head domains. Phosphorylation sites of myorod were identified, three at the C-terminal tip and three at the N-terminal domain. In the presence of calcium, addition of myosin to the TW-MR complex resulted in inhibition of this phosphorylation, while in the absence of myosin, this inhibition was negligible. Added myosin also inhibited phosphorylation of twitchin by PKA-like kinase, the latter also present in the complex. The opposite was true with the TW-MY-MR complex; that is, phosphorylation of myosin was inhibited by twitchin and/or myorod. Thus, in parallel to the well-established direct activation by calcium, molluscan catch muscle myosin also regulated its own phosphorylation. Therefore, in addition to the established phosphorylation of twitchin by PKA-like kinase, phosphorylation of myosin and myorod by myosin-associated kinase appears to play an important role in the development of the catch state.


Subject(s)
Calcium/metabolism , Calmodulin-Binding Proteins/antagonists & inhibitors , Mollusca/enzymology , Muscle, Smooth/enzymology , Myosin-Light-Chain Kinase/chemistry , Myosin-Light-Chain Kinase/metabolism , Myosins/metabolism , Animals , Calcium/chemistry , Calmodulin-Binding Proteins/metabolism , Mollusca/metabolism , Muscle Contraction , Muscle, Smooth/metabolism , Phosphorylation
14.
Cell Motil Cytoskeleton ; 64(12): 951-65, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17868135

ABSTRACT

Caldesmon (CaD), a component of microfilaments in all cells and thin filaments in smooth muscle cells, is known to bind to actin, tropomyosin, calmodulin, and myosin and to inhibit actin-activated ATP hydrolysis by smooth muscle myosin. Thus, it is believed to regulate smooth muscle contraction, cell motility and the cytoskeletal structure. Using bladder smooth muscle cell cultures and RNA interference (RNAi) technique, we show that the organization of actin into microfilaments in the cytoskeleton is diminished by siRNA-mediated CaD silencing. CaD silencing significantly decreased the amount of polymerized actin (F-actin), but the expression of actin was not altered. Additionally, we find that CaD is associated with 10 nm intermediate-sized filaments (IF) and in vitro binding assay reveals that it binds to vimentin and desmin proteins. Assembly of vimentin and desmin into IF is also affected by CaD silencing, although their expression is not significantly altered when CaD is silenced. Electronmicroscopic analyses of the siRNA-treated cells showed the presence of myosin filaments and a few surrounding actin filaments, but the distribution of microfilament bundles was sparse. Interestingly, the decrease in CaD expression had no effect on tubulin expression and distribution of microtubules in these cells. These results demonstrate that CaD is necessary for the maintenance of actin microfilaments and intermediate-sized filaments in the cytoskeletal structure. This finding raises the possibility that the cytoskeletal structure in smooth muscle is affected when CaD expression is altered, as in smooth muscle de-differentiation and hypertrophy seen in certain pathological conditions.


Subject(s)
Actin Cytoskeleton/ultrastructure , Calmodulin-Binding Proteins/physiology , Intermediate Filaments/ultrastructure , Muscle, Smooth/ultrastructure , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/metabolism , Actins/analysis , Actins/metabolism , Animals , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Cell Line , Intermediate Filaments/chemistry , Intermediate Filaments/metabolism , Muscle, Smooth/metabolism , RNA Interference , RNA, Small Interfering/pharmacology , Rabbits , Urinary Bladder/cytology
15.
Recent Pat CNS Drug Discov ; 2(2): 113-23, 2007 Jun.
Article in English | MEDLINE | ID: mdl-18221222

ABSTRACT

Neurodegenerative disease broadly includes many different diseases, such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's, dementias with Lewy bodies, post-traumatic brain injury, and stroke. Although few common physiopathological changes have been discovered among these conditions, the semiology (if known), the triggered molecular pathways that lead to the observed pathologies, and the symptomatology are essentially different. These differences entail that the treatments, both current and future, have disease-specific indications. This idea led us to believe than it would be quite impossible to comprehensively review the progress made in drug discovery for all the neurodegenerative diseases and, therefore, we focused our attention in this review on the cutting-edge patents that pertain to the treatment of Alzheimer's disease (AD). Basic science discoveries have identified new targets/leads that have led the scientific community to develop new research initiatives in order to develop novel therapeutics entities and approaches. The purpose of this review is to discuss, through cutting-edge patents, the emergence of potential future treatments of AD. We hope to provide the reader with a broader and better understanding of what could be new therapies for AD during the next decade.


Subject(s)
Alzheimer Disease/drug therapy , Drug Design , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/immunology , Amyloid beta-Protein Precursor/metabolism , Animals , Calmodulin-Binding Proteins/antagonists & inhibitors , Chelating Agents/therapeutic use , Cholinesterase Inhibitors/therapeutic use , Glycogen Synthase Kinase 3/antagonists & inhibitors , Humans , Immunization , Patents as Topic
16.
Cardiovasc Hematol Agents Med Chem ; 4(1): 61-6, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16529550

ABSTRACT

Endogenous Ouabain (EO) and Adducin enhance the Na-K pump function and play an important role in sodium homeostasis and blood pressure (BP) regulation. In the general population, plasma EO modulates BP either by inhibiting the prohypertensive effect of an excessive salt intake or counteracting the depressor action of normal-moderate salt intake. Almost 50% of hypertensive patients have increased circulating plasma levels of EO. EO has been associated both to left ventricular dysfunction and hypertrophy. A new antihypertensive agent, PST2238, (17beta-(3-furyl)-5beta-androstan-3beta, 14beta, 17alpha-triol a digitoxigenin derivative) able to selectively antagonize both the EO and adducin prohypertensive and molecular effects, has been developed. In hypertensive rats (MHS strain) carrying both adducin mutations and increased plasma EO and in ouabain-infused rats (OS), PST2238 lowers BP by normalizing the renal Na-K pump function. In OS rats, PST antagonized the cardiac and renal pro-hypertrophic ouabain effect associated to the activation of the Src-EGFr-ERK(1/2) signaling cascade. Phase 1 clinical studies demonstrated a high tolerability of PST2238. In a preliminary phase 2 study on 42 mild never-treated hypertensive patients, PST2238 given for 3 months at 0.5 mg/day, significantly reduced BP in subjects with moderate salt intake, implying that it may be selectively effective in conditions where EO plays a prohypertensive role. In conclusion, PST2238, because of its peculiar action mechanism, represents a new tool to disentangle the complex relationship between salt intake, genetic control of renal sodium handling and EO effect.


Subject(s)
Androstanols/pharmacology , Androstanols/therapeutic use , Antihypertensive Agents/pharmacology , Calmodulin-Binding Proteins/antagonists & inhibitors , Hypertension/drug therapy , Ouabain/antagonists & inhibitors , Antihypertensive Agents/therapeutic use , Blood Pressure/drug effects , Blood Pressure/physiology , Calmodulin-Binding Proteins/blood , Humans , Hypertension/physiopathology , Ouabain/blood , Ouabain/pharmacology , Risk Factors , Sodium Chloride/administration & dosage , Sodium Chloride/metabolism , Sodium-Potassium-Exchanging ATPase/drug effects , Sodium-Potassium-Exchanging ATPase/physiology
17.
Cell Mol Biol (Noisy-le-grand) ; 52(8): 15-8, 2006 Dec 30.
Article in English | MEDLINE | ID: mdl-17535730

ABSTRACT

Experimental and clinical evidence indicates that Endogenous Ouabain (EO) and Adducin polymorphism play a pathogenetic role in hypertension and related organ complications. These effects occur through a complex interaction of genetic molecular mechanisms regulating renal sodium reabsorption and vascular function. The activation of a Na-K ATPase-Src-EGFr-ERK signaling pathway within the restricted membrane subdomains of caveolae by Ouabain has been associated to hypertension and cardiac remodeling. Rostafuroxin (PST 2238) is a novel anti-hypertensive compound able to selectively antagonize EO/Ouabain and Adducin hypertensive effect and Ouabain-induced cardiac hypertrophy in rats. Studies have been conducted in vivo and in a cell-free system to prove that Rostafuroxin exerts its antihypertensive and antihypertrophic effects by antagonizing the Src-dependent signaling triggered by Ouabain. At the vascular level, Rostafuroxin antagonizes the Ouabain-mediated increase of myogenic vascular tone. This peculiar and novel mechanism of action, together with a good tolerability and efficacy both in animal models and hypertensive patients, make Rostafuroxin the prototype of a new class of antihypertensive compounds able to antagonize EO/ Ouabain and Adducin molecular effects.


Subject(s)
Androstanols/pharmacology , Antihypertensive Agents/pharmacology , Caveolae/metabolism , Hypertension/drug therapy , Ouabain/metabolism , Animals , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Caveolae/drug effects , Humans , Rats , Signal Transduction
18.
Eur J Neurosci ; 18(8): 2133-45, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14622174

ABSTRACT

We investigated the involvement of store-operated channels (SOCs) and transient receptor potential (TRP) channels in the response to activation of the group I metabotropic glutamate receptor subtype 1 (mGluR1) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG, puff application) in dopamine neurons in rat brain slices. The mGluR1-induced conductance reversed polarity close to 0 mV and at more positive potentials when extracellular potassium concentrations were increased, indicating the involvement of a cationic channel. DHPG currents but not intracellular calcium responses were reduced by low extracellular sodium concentrations but were not affected by sodium channel blockers, tetrodotoxin and saxitoxin or by inhibition of the h-current with cesium. Abolition of calcium responses with intracellular BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; 10 mm) did not affect current responses, indicating they were not calcium activated. Extracellular application of non-selective SOCs and TRP channel blockers 2-aminoethoxydiphenylborane (2-APB), SKF96365, ruthenium red and flufenamic acid (but not gadolinium) reduced DHPG current and calcium responses. Intracellular application of ruthenium red and 2-APB did not affect DHPG currents, indicating that IP3 and ryanodine receptors did not mediate their actions. Single-cell PCR revealed the presence of TRPC1 and 5 mRNA in most dopamine neurons and subtypes 3, 4 and 6 in some. Store depletion evoked calcium entry indicative of SOCs, providing the first functional observation of such channels in native central neurons. Store depletion with either cyclopiazonic acid or ryanodine abolished calcium but not current responses to DHPG. The electrophysiological and pharmacological properties of the mGluR1-induced inward current are consistent with the involvement of TRP channels whereas calcium responses are dependent on the function of SOCs in voltage clamp recordings.


Subject(s)
Calmodulin-Binding Proteins/metabolism , Dopamine/metabolism , Mesencephalon/cytology , Methoxyhydroxyphenylglycol/analogs & derivatives , Neurons/metabolism , Receptors, Metabotropic Glutamate/metabolism , Transient Receptor Potential Channels/metabolism , Animals , Animals, Newborn , Calcium Channel Blockers/pharmacology , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Female , GABA Antagonists/pharmacology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mesencephalon/drug effects , Methoxyhydroxyphenylglycol/pharmacology , Neurons/drug effects , Patch-Clamp Techniques , Rats , Rats, Wistar
19.
Cell Struct Funct ; 25(4): 243-51, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11129794

ABSTRACT

The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.


Subject(s)
Calmodulin-Binding Proteins/physiology , Calmodulin/physiology , Phagocytosis/physiology , Protozoan Proteins/physiology , Tetrahymena thermophila/physiology , Actins/metabolism , Animals , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Guinea Pigs , Peptide Elongation Factor 1/metabolism , Phagocytosis/drug effects , Protozoan Proteins/antagonists & inhibitors , Sulfonamides/pharmacology , Tetrahymena thermophila/metabolism
20.
Biochemistry ; 39(36): 11114-20, 2000 Sep 12.
Article in English | MEDLINE | ID: mdl-10998250

ABSTRACT

It is well-known that caldesmon (CaD) is a substrate for casein kinase II (CKII), and the phosphorylation of CaD by CKII regulates the interaction of CaD with myosin. However, the functionally relevant CKII phosphorylation site(s) on CaD and the precise role of CaD phosphorylation by CKII in mediating CaD's function have remained elusive. In this study, we demonstrate that Ser-26 is the major CKII phosphorylation site on CaD, while Ser-73 is of relatively minor importance. Moreover, the phosphorylation of Ser-26 and Ser-73 reduced CaD's ability to bind myosin by 45% and 27%, respectively, suggesting that the interaction of CaD with myosin is downregulated, at least in part, by the phosphorylation of these serine residues by CKII. Our results also demonstrate that there are at least four myosin-binding motifs within the amino-terminal region of CaD, located between residues 1-23, 34-43, 44-53, and 86-115, respectively. The myosin-binding motif between residues 44-53 contributes to strong myosin binding, while the three other myosin-binding motifs are responsible for weak myosin binding. The sequences between residues 24-33 and 54-85 on CaD are not required for the binding of CaD to myosin; thus, both Ser-26 and Ser-73 are located outside of the myosin-binding motifs. It is therefore likely that the downregulation of myosin-CaD interactions by CKII phosphorylation is due to phosphorylation-induced conformational changes in the adjacent myosin-binding motifs on CaD, rather than by the direct modification of these myosin-binding motifs by CKII.


Subject(s)
Calmodulin-Binding Proteins/metabolism , Down-Regulation , Myosins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Binding Sites/genetics , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/genetics , Casein Kinase II , Chickens , Down-Regulation/genetics , Muscle, Smooth/metabolism , Mutagenesis, Site-Directed , Myosins/antagonists & inhibitors , Phosphorylation , Point Mutation , Protein Binding/genetics , Sequence Deletion , Serine/genetics , Structure-Activity Relationship , Threonine/genetics
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