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1.
Nat Commun ; 12(1): 1955, 2021 03 29.
Article in English | MEDLINE | ID: mdl-33782410

ABSTRACT

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.


Subject(s)
Autophagy/genetics , Cell Cycle Proteins/genetics , Molecular Chaperones/genetics , Protein Aggregates , Sequestosome-1 Protein/genetics , Superoxide Dismutase-1/genetics , Animals , Autophagy/drug effects , Cell Cycle Proteins/metabolism , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Leupeptins/pharmacology , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Chaperones/metabolism , Motor Neurons/cytology , Motor Neurons/drug effects , Motor Neurons/metabolism , Primary Cell Culture , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/metabolism , Protein Aggregates/drug effects , Protein Folding/drug effects , Sequestosome-1 Protein/metabolism , Signal Transduction , Superoxide Dismutase-1/metabolism , Transgenes , Xenopus laevis , Red Fluorescent Protein
2.
J Exp Clin Cancer Res ; 38(1): 363, 2019 Aug 19.
Article in English | MEDLINE | ID: mdl-31426843

ABSTRACT

BACKGROUND: In order to develop a new immunotherapeutic agent targeting metastatic breast cancers, we chose to utilize autocatalytic feature of the membrane serine protease Prss14/ST14, a specific prognosis marker for ER negative breast cancer as a target molecule. METHODS: The study was conducted using three mouse breast cancer models, 4 T1 and E0771 mouse breast cancer cells into their syngeneic hosts, and an MMTV-PyMT transgenic mouse strain was used. Prss14/ST14 knockdown cells were used to test function in tumor growth and metastasis, peptides derived from the autocatalytic loop for activation were tested as preventive metastasis vaccine, and monoclonal and humanized antibodies to the same epitope were tested as new therapeutic candidates. ELISA, immunoprecipitation, Immunofluorescent staining, and flow cytometry were used to examine antigen binding. The functions of antibodies were tested in vitro for cell migration and in vivo for tumor growth and metastasis. RESULTS: Prss14/ST14 is critically involved in the metastasis of breast cancer and poor survival rather than primary tumor growth in two mouse models. The epitopes derived from the specific autocatalytic loop region of Prss14/ST14, based on structural modeling acted as efficient preventive metastasis vaccines in mice. A new specific monoclonal antibody mAb3F3 generated against the engineered loop structure could reduce cell migration, eliminate metastasis in PyMT mice, and can detect the Prss14/ST14 protein expressed in various human cancer cells. Humanized antibody huAb3F3 maintained the specificity and reduced the migration of human breast cancer cells in vitro. CONCLUSION: Our study demonstrates that Prss14/ST14 is an important target for modulating metastasis. Our newly developed hybridoma mAbs and humanized antibody can be further developed as new promising candidates for the use in diagnosis and in immunotherapy of human metastatic breast cancer.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal/pharmacology , Breast Neoplasms/prevention & control , Epitopes/immunology , Lung Neoplasms/prevention & control , Peptide Fragments/immunology , Serine Endopeptidases/immunology , Animals , Antigens, Polyomavirus Transforming/genetics , Apoptosis , Breast Neoplasms/immunology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle , Cell Movement , Cell Proliferation , Female , Humans , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
3.
J Biol Chem ; 292(24): 9858-9864, 2017 06 16.
Article in English | MEDLINE | ID: mdl-28487468

ABSTRACT

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.


Subject(s)
Antioxidants/metabolism , Catechin/analogs & derivatives , ErbB Receptors/metabolism , Integrin beta3/metabolism , Lipid Bilayers/metabolism , Models, Molecular , Signal Transduction , Amino Acid Substitution , Animals , Antioxidants/chemistry , Antioxidants/therapeutic use , CHO Cells , Catechin/chemistry , Catechin/metabolism , Catechin/therapeutic use , Cricetulus , Dietary Supplements , Dimerization , ErbB Receptors/agonists , ErbB Receptors/chemistry , ErbB Receptors/genetics , Humans , Integrin alpha2/chemistry , Integrin alpha2/genetics , Integrin alpha2/metabolism , Integrin beta3/chemistry , Integrin beta3/genetics , Ligands , Lipid Bilayers/chemistry , Mutation , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/agonists , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Talin/antagonists & inhibitors , Talin/chemistry , Talin/metabolism
4.
Chem Biol Drug Des ; 90(5): 873-882, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28432753

ABSTRACT

Cardiovascular disease, which is caused by unregulated platelet aggregation, is one of the main causes of deaths worldwide. Many studies have focused on natural products with antiplatelet effects as a safe alternative therapy to prevent the disease. In this context, an in-house chemical library was screened to find natural products capable of inhibiting the interaction between platelet integrin αIIbß3 and fibrinogen, which is an essential step in platelet aggregation. On the basis of the screening results, indothiazinone, an alkaloid found in microbial cultures, was identified as a potential antiplatelet agent. Specifically, indothiazinone treatment significantly inhibited the binding of fibrinogen to Chinese hamster ovary cells expressing integrin αIIbß3. It also restricted thrombin- and adenosine diphosphate-dependent spreading of human platelets on a fibrinogen matrix. More importantly, surface plasmon resonance and molecular dynamics studies suggested that indothiazinone suppressed talin-induced activation of integrin αIIbß3 presumably by inhibiting talin-integrin interaction. In conclusion, these results suggest that indothiazinone can be used as a lead compound for the development of antiplatelet drugs with a novel mode of action.


Subject(s)
Blood Platelets/drug effects , Indoles/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Thiazoles/pharmacology , Animals , Blood Platelets/cytology , Blood Platelets/metabolism , CHO Cells , Cricetulus , Humans , Indoles/chemistry , Models, Molecular , Platelet Aggregation Inhibitors/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Talin/metabolism , Thiazoles/chemistry
5.
FEBS Lett ; 590(20): 3517-3525, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27658040

ABSTRACT

Regulation of integrin affinity for its ligand is essential for cell adhesion and migration. Here, we found that direct interaction of vimentin with integrin ß1 can enhance binding of integrin α5ß1 to its ligand, fibronectin. Conversely, blocking the interaction reduced fibronectin binding, cell migration on a fibronectin-coated surface, and neural tube closure during Xenopus embryogenesis. We also found that withaferin A (WFA), a natural compound known to inhibit vimentin function, can suppress the vimentin-integrin interaction and abolish fibronectin binding. Finally, we identified Ser38 of vimentin as a critical residue for integrin binding. Our results suggest that phosphorylation of vimentin at Ser38 may regulate the integrin-ligand interaction, thus providing a molecular basis for antivimentin therapeutic strategies.


Subject(s)
Integrin alpha5beta1/metabolism , Integrin beta1/metabolism , Serine/metabolism , Vimentin/chemistry , Vimentin/metabolism , Xenopus/embryology , Animals , Binding Sites/drug effects , CHO Cells , Cell Adhesion , Cell Movement/drug effects , Cricetulus , Fibronectins/metabolism , Integrin alpha5beta1/chemistry , Integrin beta1/chemistry , Neural Tube/metabolism , Protein Binding/drug effects , Withanolides/pharmacology , Xenopus/metabolism , Xenopus Proteins/chemistry , Xenopus Proteins/metabolism
6.
Arterioscler Thromb Vasc Biol ; 36(7): 1406-16, 2016 07.
Article in English | MEDLINE | ID: mdl-27199448

ABSTRACT

OBJECTIVE: Angiogenesis, the process of building complex vascular structures, begins with sprout formation on preexisting blood vessels, followed by extension of the vessels through proliferation and migration of endothelial cells. Based on the potential therapeutic benefits of preventing angiogenesis in pathological conditions, many studies have focused on the mechanisms of its initiation as well as control. However, how the extension of vessels is terminated remains obscure. Thus, we investigated the negative regulation mechanism. APPROACH AND RESULTS: We report that increased intracellular calcium can induce dephosphorylation of the endothelial receptor tyrosine kinase Tie2. The calcium-mediated dephosphorylation was found to be dependent on Tie2-calmodulin interaction. The Tyr1113 residue in the C-terminal end loop of the Tie2 kinase domain was mapped and found to be required for this interaction. Moreover, mutation of this residue into Phe impaired both the Tie2-calmodulin interaction and calcium-mediated Tie2 dephosphorylation. Furthermore, expressing a mutant Tie2 incapable of binding to calmodulin or inhibiting calmodulin function in vivo causes unchecked growth of the vasculature in Xenopus. Specifically, knockdown of Tie2 in Xenopus embryo retarded the sprouting and extension of intersomitic veins. Although human Tie2 expression in the Tie2-deficient animals almost completely rescued the retardation, the Tie2(Y1113F) mutant caused overgrowth of intersomitic veins with strikingly complex and excessive branching patterns. CONCLUSIONS: We propose that the calcium/calmodulin-dependent negative regulation of Tie2 can be used as an inhibitory signal for vessel growth and branching to build proper vessel architecture during embryonic development.


Subject(s)
Calcium Signaling , Calmodulin/metabolism , Embryo, Nonmammalian/blood supply , Human Umbilical Vein Endothelial Cells/metabolism , Neovascularization, Physiologic , Receptor, TIE-2/metabolism , Xenopus/embryology , Animals , CHO Cells , Cricetulus , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Genotype , HEK293 Cells , Humans , Mutation , Phenotype , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Receptor, TIE-2/genetics , Transfection , Tyrosine , Xenopus/genetics , Xenopus/metabolism
7.
J Cell Sci ; 129(10): 2030-42, 2016 05 15.
Article in English | MEDLINE | ID: mdl-27044755

ABSTRACT

Vimentin, an intermediate filament protein induced during epithelial-to-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin ß3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin ß3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin ß3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin-ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin-integrin interaction might control cell adhesion.


Subject(s)
Cell Adhesion/genetics , Cytoskeleton/metabolism , Integrin beta3/genetics , Vimentin/genetics , Animals , CHO Cells , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Movement/genetics , Cricetinae , Cricetulus , Cytoskeleton/genetics , Epithelial-Mesenchymal Transition/genetics , Humans , Integrin beta3/metabolism , Ligands , Protein Binding , Protein Interaction Maps , Proteomics , Vimentin/metabolism
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