scholarly journals Mechanisms of Kinesin-1 activation by Ensconsin/MAP7 in vivo

2018 ◽  
Author(s):  
Mathieu Métivier ◽  
Brigette Y. Monroy ◽  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
...  
Keyword(s):  
Large Cell ◽  
Binding Domain ◽  
Microtubule Network ◽  
Centrosome Separation ◽  
Microtubule Binding Domain ◽  
Dual Activation ◽  
First Time ◽  
Dependent Processes

AbstractCentrosome separation in Drosophila larval neuroblasts and asymmetric transport of embryonic determinants in oocytes are both microtubule-dependent processes that require Kinesin-1 activation by Ensconsin/microtubule-associated protein 7 (MAP7). However, the molecular mechanism used by Ensconsin to activate Kinesin-1 remains elusive. Ensconsin/ MAP7 contains an N-terminal microtubule-binding domain (MBD) and a C-terminal Kinesin-binding domain (KBD). Using rescue experiments in live flies, we show that KBD expression alone is sufficient to fully rescue Ensconsin-dependent centrosome separation defects, but not the fast oocyte streaming and the localization patterns of Staufen and Gurken proteins. Interestingly, we show here for the first time that KBD binds and stimulates Kinesin-1 binding to Mts in vivo and in vitro. We propose that the KBD/Kinesin-1 motor represents a minimal activation module that stimulates Kinesin-1 binding to Mts. Addition of the MBD, present in the full length Ensconsin allows this activation to occur directly on the Mt. Our data also suggest that in a very large cell with a complex microtubule network, but not in smaller cells, this dual activation by Ensconsin is essential for optimal Kinesin-1 targeting to the microtubule cytoskeleton.

scholarly journals Modulation of tau phosphorylation and intracellular localization by cellular stress

2000 ◽  
Vol 345 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Scott M. JENKINS ◽  
Marcus ZINNERMAN ◽  
Craig GARNER ◽  
Gail V. W. JOHNSON
Keyword(s):  
Osmotic Stress ◽  
Protein Kinases ◽  
Intracellular Localization ◽  
Tau Phosphorylation ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Microtubule Binding Domain

Tau is a microtubule-associated protein that is functionally modulated by phosphorylation and hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of great interest to identify the signalling pathways and enzymes capable of modulating tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and localization in response to osmotic stress, which activates the stress-activated protein kinases (SAPKs), a family of proline-directed protein kinases shown to phosphorylate tau in vitro and hypothesized to phosphorylate tau in Alzheimer's disease. Immunoblot analysis with phosphorylation-dependent antibodies revealed that osmotic stress increased tau phosphorylation at the non-Ser/Thr-Pro sites Ser-262/356, within the microtubule-binding domain, as well as Ser/Thr-Pro sites outside of tau's microtubule-binding domain. Although all SAPKs examined were activated by osmotic stress, none of the endogenous SAPKs mediated the increase in tau phosphorylation. However, when transfected into SH-SY5Y cells, SAPK3, but not the other SAPKs examined, phosphorylated tau in situ in response to activation by osmotic stress. Osmotic-stress-induced tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and an increase in the amount of soluble tau. This stress-induced alteration in tau localization was only partially due to phosphorylation at Ser-262/356 by a staurosporine-sensitive, non-proline-directed, protein kinase. Taken together, these results suggest that osmotic stress activates at least two tau-directed protein kinases, one proline-directed and one non-proline-directed, that SAPK3 can phosphorylate tau on Ser/Thr-Pro residues in situ, and that Ser-262/356 phosphorylation only partially regulates tau localization in the cell.

Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins

2001 ◽  
Vol 114 (1) ◽  
pp. 161-172 ◽  
Author(s):  
D. Sun ◽  
C.L. Leung ◽  
R.K. Liem
Keyword(s):  
Calcium Binding ◽  
Binding Domain ◽  
Actin Binding ◽  
Microtubule Binding ◽  
Transfected Cells ◽  
Terminal Domain ◽  
Microtubule Binding Domain

MACF (microtubule actin cross-linking factor) is a large, 608-kDa protein that can associate with both actin microfilaments and microtubules (MTs). Structurally, MACF can be divided into 3 domains: an N-terminal domain that contains both a calponin type actin-binding domain and a plakin domain; a rod domain that is composed of 23 dystrophin-like spectrin repeats; and a C-terminal domain that includes two EF-hand calcium-binding motifs, as well as a region that is homologous to two related proteins, GAR22 and Gas2. We have previously demonstrated that the C-terminal domain of MACF binds to MTs, although no homology was observed between this domain and other known microtubule-binding proteins. In this report, we describe the characterization of this microtubule-binding domain of MACF by transient transfection studies and in vitro binding assays. We found that the C-terminus of MACF contains at least two microtubule-binding regions, a GAR domain and a domain containing glycine-serine-arginine (GSR) repeats. In transfected cells, the GAR domain bound to and partially stabilized MTs to depolymerization by nocodazole. The GSR-containing domain caused MTs to form bundles that are still sensitive to nocodazole-induced depolymerization. When present together, these two domains acted in concert to bundle MTs and render them stable to nocodazole treatment. Recently, a study has shown that the N-terminal half of the plakin domain (called the M1 domain) of MACF also binds MTs. We therefore examined the microtubule binding ability of the M1 domain in the context of the entire plakin domain with and without the remaining N-terminal regions of two different MACF isoforms. Interestingly, in the presence of the surrounding sequences, the M1 domain did not bind MTs. In addition to MACF, cDNA sequences encoding the GAR and GSR-containing domains are also found in the partial human EST clone KIAA0728, which has high sequence homology to the 3′ end of the MACF cDNA; hence, we refer to it as MACF2. The C-terminal domain of mouse MACF2 was cloned and characterized. The microtubule-binding properties of MACF2 C-terminal domain are similar to that of MACF. The GAR domain was originally found in Gas 2 protein and here we show that it can associate with MTs in transfected cells. Plectin and desmoplakin have GSR-containing domains at their C-termini and we further demonstrate that the GSR-containing domain of plectin, but not desmoplakin, can bind to MTs in vivo.

ALK+ALCLs induce cutaneous, HMGB-1–dependent IL-8/CXCL8 production by keratinocytes through NF-κB activation

Blood ◽  
2012 ◽  
Vol 119 (20) ◽  
pp. 4698-4707 ◽  
Author(s):  
Emilie Dejean ◽  
Marianne Foisseau ◽  
Fréderic Lagarrigue ◽  
Laurence Lamant ◽  
Naïs Prade ◽  
...  
Keyword(s):  
High Mobility ◽  
Large Cell ◽  
Premetastatic Niche ◽  
Anaplastic Lymphoma ◽  
Number Of Patients ◽  
Skin Colonization ◽  
Cell Invasiveness ◽  
First Time

Abstract Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK+ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase–positive (ALK+) lymphomas and human ALK+ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK+ cells, and demonstrate extracellular HMGB-1–stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK+ cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK+ cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK+ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK+ cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK+ lymphoma epidermotropism.

Phosphorylation of MAP4 affects microtubule properties and cell cycle progression

2001 ◽  
Vol 114 (15) ◽  
pp. 2879-2887 ◽  
Author(s):  
Winston Chang ◽  
Dorota Gruber ◽  
Sripriya Chari ◽  
Hidefumi Kitazawa ◽  
Yuko Hamazumi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Binding Domain ◽  
Proliferating Cells ◽  
Cycle Progression ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
In Cells

In human cells, MAP4, a microtubule-associated protein ubiquitously expressed in proliferating cells, has been shown to undergo in vivo phosphorylation. Two phosphorylation sites, serines 696 and 787, lie within the proline-rich region of its microtubule-binding domain. To test the hypothesis that phosphorylation at these sites influences microtubule properties or cell cycle progression, we prepared stable cell lines that inducibly express versions of MAP4 in which phosphorylation of these two serines was prevented by their replacement with alanine, lysine, or glutamate residues (AA-, KK-, or EE-MAP4). All non-phosphorylatable mutant forms of MAP4 expressed in mouse Ltk- cells were localized to MT arrays that were unremarkable in appearance. Expression of non-phosphorylatable mutants of MAP4 did not affect cell doubling time; however, expression of some mutants altered progression into or through cell division. Interactions of mutant MAP4 with MTs were examined in vitro. KK mutant MAP4 bound MTs more avidly than its wild-type counterpart, WT-MAP4. In vivo MT polymer also differed among the mutants: MTs in cells expressing the KK- and AA-MAP4 forms were more resistant to nocodazole depolymerization than those in cells expressing EE- or WT-MAP4 forms. Our results demonstrate that phosphorylation alters MAP4 properties and suggest a raison d'être for phosphorylation of the MAP4 microtubule-binding domain during cell cycle progression.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Fe3O4@Pt nanoparticles to enable combinational electrodynamic/chemodynamic therapy

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Tong Chen ◽  
Qiang Chu ◽  
Mengyang Li ◽  
Gaorong Han ◽  
Xiang Li
Keyword(s):  
Fenton Reaction ◽  
Pt Nanoparticles ◽  
Therapeutic Modality ◽  
Ros Generation ◽  
Tumor Treatment ◽  
Superior Efficacy ◽  
Platinum Nanocrystals ◽  
First Time

AbstractElectrodynamic therapy (EDT) has recently emerged as a potential external field responsive approach for tumor treatment. While it presents a number of clear superiorities, EDT inherits the intrinsic challenges of current reactive oxygen species (ROS) based therapeutic treatments owing to the complex tumor microenvironment, including glutathione (GSH) overexpression, acidity and others. Herein for the first time, iron oxide nanoparticles are decorated using platinum nanocrystals (Fe3O4@Pt NPs) to integrate the current EDT with chemodynamic phenomenon and GSH depletion. Fe3O4@Pt NPs can effectively induce ROS generation based on the catalytic reaction on the surface of Pt nanoparticles triggered by electric field (E), and meanwhile it may catalyze intracellular H2O2 into ROS via Fenton reaction. In addition, Fe3+ ions released from Fe3O4@Pt NPs under the acidic condition in tumor cells consume GSH in a rapid fashion, inhibiting ROS clearance to enhance its antitumor efficacy. As a result, considerable in vitro and in vivo tumor inhibition phenomena are observed. This study has demonstrated an alternative concept of combinational therapeutic modality with superior efficacy.

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 Schistosoma mansoni eggs induce Wnt/β-catenin signaling and activate the protooncogene c-Jun in human and hamster colon

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jakob Weglage ◽  
Friederike Wolters ◽  
Laura Hehr ◽  
Jakob Lichtenberger ◽  
Celina Wulz ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Colorectal Carcinogenesis ◽  
Sub Saharan Africa ◽  
Interleukin 4 ◽  
Health Burden ◽  
Sub Saharan ◽  
Considerable Morbidity ◽  
First Time

AbstractSchistosomiasis (bilharzia) is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, with considerable morbidity in parts of the Middle East, South America, Southeast Asia, in sub-Saharan Africa, and particularly also in Europe. The WHO describes an increasing global health burden with more than 290 million people threatened by the disease and a potential to spread into regions with temperate climates like Corsica, France. The aim of our study was to investigate the influence of S. mansoni infection on colorectal carcinogenic signaling pathways in vivo and in vitro. S. mansoni infection, soluble egg antigens (SEA) and the Interleukin-4-inducing principle from S. mansoni eggs induce Wnt/β-catenin signaling and the protooncogene c-Jun as well as downstream factor Cyclin D1 and markers for DNA-damage, such as Parp1 and γH2a.x in enterocytes. The presence of these characteristic hallmarks of colorectal carcinogenesis was confirmed in colon biopsies from S. mansoni-infected patients demonstrating the clinical relevance of our findings. For the first time it was shown that S. mansoni SEA may be involved in the induction of colorectal carcinoma-associated signaling pathways.

scholarly journals AZP2006, a new promising treatment for Alzheimer’s and related diseases

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
N. Callizot ◽  
C. Estrella ◽  
S. Burlet ◽  
A. Henriques ◽  
C. Brantis ◽  
...  
Keyword(s):  
Therapeutic Application ◽  
Neuroprotective Effects ◽  
Beneficial Effects ◽  
Potential Therapeutic Application ◽  
Promising Treatment ◽  
Central Synapses ◽  
First Time ◽  
Pgrn Level

AbstractProgranulin (PGRN) is a protein with multiple functions including the regulation of neuroinflammation, neuronal survival, neurite and synapsis growth. Although the mechanisms of action of PGRN are currently unknown, its potential therapeutic application in treating neurodegenerative diseases is huge. Thus, strategies to increase PGRN levels in patients could provide an effective treatment. In the present study, we investigated the effects of AZP2006, a lysotropic molecule now in phase 2a clinical trial in Progressive Supranuclear Palsy patients, for its ability to increase PGRN level and promote neuroprotection. We showed for the first time the in vitro and in vivo neuroprotective effects of AZP2006 in neurons injured with Aβ1–42 and in two different pathological animal models of Alzheimer’s disease (AD) and aging. Thus, the chronic treatment with AZP2006 was shown to reduce the loss of central synapses and neurons but also to dramatically decrease the massive neuroinflammation associated with the animal pathology. A deeper investigation showed that the beneficial effects of AZP2006 were associated with PGRN production. Also, AZP2006 binds to PSAP (the cofactor of PGRN) and inhibits TLR9 receptors normally responsible for proinflammation when activated. Altogether, these results showed the high potential of AZP2006 as a new putative treatment for AD and related diseases.

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