scholarly journals UNC-45A Weakens and Breaks MT Lattice Independent of its Effect on Non-Muscle Myosin II

2020 ◽  
Author(s):  
Juri Habicht ◽  
Ashley Mooneyham ◽  
Asumi Hoshino ◽  
Mihir Shetty ◽  
Xiaonan Zhang ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Myosin Ii ◽  
Human Diseases ◽  
Therapeutic Implications ◽  
Associated Functions ◽  
Muscle Myosin ◽  
First Time ◽  
In Cells

AbstractIn invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show for the first time that: a) in vitro UNC-45A binds to the MT lattice and weakens its integrity leading to MT bending, breakage and depolymerization, b) in cells, UNC-45A overexpression causes loss of MT mass and increase in MT breakages, c) both in vitro and in cells, UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and destabilization occurs even in presence of the NMII inhibitor blebbistatin. These findings are consistent with a not mutually exclusive but rather dual role of UNC-45A in regulating NMII activity and MT stability.Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.

scholarly journals UNC-45A breaks the microtubule lattice independently of its effects on non-muscle myosin II

2020 ◽  
Vol 134 (1) ◽  
pp. jcs248815
Author(s):  
Juri Habicht ◽  
Ashley Mooneyham ◽  
Asumi Hoshino ◽  
Mihir Shetty ◽  
Xiaonan Zhang ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Myosin Ii ◽  
Muscle Cells ◽  
Binding Domain ◽  
Human Diseases ◽  
First Person ◽  
Therapeutic Implications ◽  
Associated Functions ◽  
Muscle Myosin

ABSTRACTIn invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only, and UNC-45A, expressed in all cells and implicated in regulating both non-muscle myosin II (NMII)- and microtubule (MT)-associated functions. Here, we show that, in vitro and in human and rat cells, UNC-45A binds to the MT lattice, leading to MT bending, breakage and depolymerization. Furthermore, we show that UNC-45A destabilizes MTs independent of its C-terminal NMII-binding domain and even in the presence of the NMII inhibitor blebbistatin. These findings identified UNC-45A as a novel type of MT-severing protein with a dual non-mutually exclusive role in regulating NMII activity and MT stability. Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability, our findings have profound implications in the biology of MTs, as well as the biology of human diseases and possible therapeutic implications for their treatment.This article has an associated First Person interview with the joint first authors of the paper.

scholarly journals The Central Role of the Tail in Switching Off Myosin II in Cells

2019 ◽  
Author(s):  
Shixin Yang ◽  
Kyoung Hwan Lee ◽  
John L. Woodhead ◽  
Osamu Sato ◽  
Mitsuo Ikebe ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Myosin Ii ◽  
Actin Binding ◽  
Compact Structure ◽  
Mechanism Of Inhibition ◽  
Energy Conserving ◽  
High Degree ◽  
First Time ◽  
In Cells

AbstractMyosin II is a motor protein playing an essential role in cell motility. The molecule can exist as a polymer that pulls on actin to generate motion, or as an inactive monomer with a compact structure, in which its tail is folded and its two heads interact with each other. This conformation functions in cells as an energy-conserving storage and transport molecule. The mechanism of inhibition is not fully understood. We have carried out a 3D reconstruction of the switched-off form revealing for the first time multiple interactions between the tail and the two heads that trap ATP hydrolysis products, block actin binding, obstruct head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function can explain the high degree of inhibition of the folded form of myosin, serving its energy-conserving, storage function in cells. The structure also suggests a mechanism for unfolding when activated by phosphorylation.

scholarly journals Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Miao-Miao Zhao ◽  
Wei-Li Yang ◽  
Fang-Yuan Yang ◽  
Li Zhang ◽  
Wei-Jin Huang ◽  
...  
Keyword(s):  
Drug Development ◽  
Cathepsin L ◽  
Human Cells ◽  
New Drugs ◽  
Disease Course ◽  
Promising Target ◽  
First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

scholarly journals Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Zhang ◽  
Guoyu Yin ◽  
Heping Zhao ◽  
Hanzhi Ling ◽  
Zhen Xie ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Hyaluronic Acid ◽  
Dependent Manner ◽  
Collagen Induced Arthritis ◽  
Intracellular Degradation ◽  
Main Pathway ◽  
First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

scholarly journals Arginase Activity in Eisenia andrei Coelomocytes: Function in the Earthworm Innate Response

2021 ◽  
Vol 22 (7) ◽  
pp. 3687
Author(s):  
Joanna Homa ◽  
Alina Klosowska ◽  
Magdalena Chadzinska
Keyword(s):  
Immune Response ◽  
Wound Healing ◽  
Arginase Activity ◽  
Eisenia Andrei ◽  
Heavy Metals Ions ◽  
First Time ◽  
Important Marker

Arginase is the manganese metalloenzyme catalyzing the conversion of l-arginine to l-ornithine and urea. In vertebrates, arginase is involved in the immune response, tissue regeneration, and wound healing and is an important marker of alternative anti-inflammatory polarization of macrophages. In invertebrates, data concerning the role of arginase in these processes are very limited. Therefore, in the present study, we focused on the changes in arginase activity in the coelomocytes of Eisenia andrei. We studied the effects of lipopolysaccharide (LPS), hydrogen peroxide (H2O2), heavy metals ions (e.g., Mn2+), parasite infection, wound healing, and short-term fasting (5 days) on arginase activity. For the first time in earthworms, we described arginase activity in the coelomocytes and found that it can be up-regulated upon in vitro stimulation with LPS and H2O2 and in the presence of Mn2+ ions. Moreover, arginase activity was also up-regulated in animals in vivo infected with nematodes or experiencing segment amputation, but not in fasting earthworms. Furthermore, we confirmed that the activity of coelomocyte arginase can be suppressed by l-norvaline. Our studies strongly suggest that similarly to the vertebrates, also in the earthworms, coelomocyte arginase is an important element of the immune response and wound healing processes.

scholarly journals Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

2021 ◽  
Vol 22 (3) ◽  
pp. 1163
Author(s):  
Gaia Palmini ◽  
Cecilia Romagnoli ◽  
Simone Donati ◽  
Roberto Zonefrati ◽  
Gianna Galli ◽  
...  
Keyword(s):  
Cell Line ◽  
Molecular Mechanisms ◽  
Osteogenic Sarcoma ◽  
Cancer Cell Line ◽  
Microscopic Features ◽  
In Vitro Establishment ◽  
Profile Characteristic ◽  
First Time

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

scholarly journals Oncogenic UBE3C promotes breast cancer progression by activating Wnt/β-catenin signaling

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chen Hang ◽  
Shanojie Zhao ◽  
Tiejun Wang ◽  
Yan Zhang
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
Nuclear Accumulation ◽  
Migration And Invasion ◽  
Ubiquitin Protein Ligase ◽  
Novel Target ◽  
First Time ◽  
Breast Tissues

Abstract Background Breast cancer (BrCa) is the most common female malignancy worldwide and has the highest morbidity among all cancers in females. Unfortunately, the mechanisms of BrCa growth and metastasis, which lead to a poor prognosis in BrCa patients, have not been well characterized. Methods Immunohistochemistry (IHC) was performed on a BrCa tissue microarray (TMA) containing 80 samples to evaluate ubiquitin protein ligase E3C (UBE3C) expression. In addition, a series of cellular experiments were conducted to reveal the role of UBE3C in BrCa. Results In this research, we identified UBE3C as an oncogenic factor in BrCa growth and metastasis for the first time. UBE3C expression was upregulated in BrCa tissues compared with adjacent breast tissues. BrCa patients with high nuclear UBE3C expression in tumors showed remarkably worse overall survival (OS) than those with low nuclear expression. Knockdown of UBE3C expression in MCF-7 and MDA-MB-453 BrCa cells inhibited cell proliferation, migration and invasion in vitro, while overexpression of UBE3C in these cells exerted the opposite effects. Moreover, UBE3C promoted β-catenin nuclear accumulation, leading to the activation of the Wnt/β-catenin signaling pathway in BrCa cells. Conclusion Collectively, these results imply that UBE3C plays crucial roles in BrCa development and progression and that UBE3C may be a novel target for the prevention and treatment of BrCa.

scholarly journals Phenotypic Susceptibility to Bevirimat in Isolates from HIV-1-Infected Patients without Prior Exposure to Bevirimat

2010 ◽  
Vol 54 (6) ◽  
pp. 2345-2353 ◽  
Author(s):  
Nicolas A. Margot ◽  
Craig S. Gibbs ◽  
Michael D. Miller
Keyword(s):  
Recombinant Viruses ◽  
Gag Polyprotein ◽  
New Class ◽  
Major Mutation ◽  
Hiv Drugs ◽  
The Impact ◽  
First Time ◽  
Hiv 1

ABSTRACT Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

scholarly journals DNAJB6b is Downregulated in Synucleinopathies

2021 ◽  
pp. 1-13
Author(s):  
Jonas Folke ◽  
Sertan Arkan ◽  
Isak Martinsson ◽  
Susana Aznar ◽  
Gunnar Gouras ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Substantia Nigra Pars Compacta ◽  
Endogenous Protein ◽  
Highly Sensitive ◽  
Brain Material ◽  
Health And Disease ◽  
Isoform B

Background: α-synuclein (α-syn) aggregation contributes to the progression of multiple neurodegenerative diseases. We recently found that the isoform b of the co-chaperone DNAJB6 is a strong suppressor of a-syn aggregation in vivo and in vitro. However, nothing is known about the role of the endogenous isoform b of DNAJB6 (DNAJB6b) in health and disease, due to lack of specific antibodies. Objective: Here we generated a novel anti-DNAJB6b antibody to analyze the localization and expression this isoform in cells, in tissue and in clinical material. Methods: To address this we used immunocytochemistry, immunohistochemistry, as well as a novel quantitative DNAJB6 specific ELISA method. Results: The endogenous protein is mainly expressed in the cytoplasm and in neurites in vitro, where it is found more in dendrites than in axons. We further verified in vivo that DNAJB6b is expressed in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), which is a neuronal subpopulation highly sensitive to α-syn aggregation, that degenerate to a large extend in patients with Parkinson’s disease (PD) and multiple system atrophy (MSA). When we analyzed the expression levels of DNAJB6b in brain material from PD and MSA patients, we found a downregulation of DNAJB6b by use of ELISA based quantification. Interestingly, this was also true when analyzing tissue from patients with progressive supranuclear palsy, a taupathic atypical parkinsonian disorder. However, the total level of DNAJB6 was upregulated in these three diseases, which may indicate an upregulation of the other major isoform of DNAJB6, DNAJB6a. Conclusion: This study shows that DNAJB6b is downregulated in several different neurodegenerative diseases, which makes it an interesting target to further investigate in relation to amyloid protein aggregation and disease progression.

Abstract 18060: TRAIL Promotes Angiogenesis and Ischemia-Induced Neovascularisation via NOX4, H2O2 and Nitric Oxide-Dependent Mechanisms

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Belinda A Di Bartolo ◽  
Sian P Cartland ◽  
Leonel Prado-Lourenco ◽  
Nor Saadah M Azahri ◽  
Thuan Thai ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Angiogenic Factor ◽  
Tubule Formation ◽  
Nitric Oxide Synthase Inhibitor ◽  
Therapeutic Implications ◽  
Synthase Inhibitor ◽  
Oxide Synthase ◽  
First Time

Background: Angiogenesis and neovascularization are essential processes that follow ischemia insults. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) not only induces endothelial cell (EC) death and inhibits angiogenesis, but also promotes EC migration, invasion and proliferation in vitro . These seemingly opposite effects make its role in angiogenesis in vivo unclear. Using TRAIL -/- and wild-type mice, we sought to determine the role of TRAIL in angiogenesis and neovascularisation. We also sought mechanisms in vitro . Methods and Results: Reduced vascularisation assessed by real-time in vivo 3D Vevo ultrasound imaging and CD31 staining was observed in TRAIL -/- mice 28 d after hindlimb ischemia. Moreover, reduced capillary formation and increased apoptosis was evident in TRAIL -/- muscles even at 3 d after ischemic surgery. We have previously shown that fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, regulates TRAIL gene expression in vascular smooth muscle cells. Indeed, FGF-2 also regulates TRAIL expression in ECs, and FGF-2-inducible proliferation, migration and tubule formation was inhibited with siRNA targeting TRAIL. Notably, both FGF-2 and TRAIL significantly increased NOX4 expression. TRAIL-inducible angiogenic activity in ECs was inhibited with siRNAs targeting NOX4, and consistent with these, NOX4 mRNA was reduced in 3 d ischemic hindlimbs of TRAIL -/- mice. TRAIL stimulated intracellular H 2 O 2 levels in ECs, and TRAIL-inducible proliferation, migration and tubule formation was inhibited with not only PEG-catalase, a H 2 O 2 scavenger, but also blocked with L-NAME, a nitric oxide synthase inhibitor. Conclusions: This is the first demonstration showing that TRAIL promotes angiogenesis in vivo . We show for the first time that the TRAIL stimulates NOX4 expression to mediate nitric oxide-dependent angiogenic effects. This has significant therapeutic implications such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with CVD and diabetes.

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