scholarly journals Reconstitution of Kinesin-1 Activation

2021 ◽  
Author(s):  
Kyoko Chiba ◽  
Kassandra M. Ori-McKenney ◽  
Shinsuke Niwa ◽  
Richard J. McKenney
Keyword(s):  
Conformational Changes ◽  
Regulatory Mechanism ◽  
Adaptor Proteins ◽  
Intramolecular Interactions ◽  
Motor Complex ◽  
In Vitro Reconstitution ◽  
Independent Mechanism ◽  
Microtubule Motor ◽  
Motor Interaction

AbstractAutoinhibition is an important regulatory mechanism for cytoskeletal motor proteins. Kinesin-1 (kinesin hereafter), the ubiquitous plus-end directed microtubule motor, is thought to be controlled by a complicated autoinihibition mechanism, but the molecular details remain unclear. Conformational changes mediated by intramolecular interactions between the C-terminal tail and N-terminal motor domains of the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The dimeric KHC also binds two copies of the kinesin light chains (KLCs), which have been implicated in both autoinhibition and cargo-dependent activation of the tetrameric motor complex, although the precise mechanisms remain opaque. Using in vitro reconstitution, we show that the KLC strongly inhibits the kinesin-microtubule interaction via an independent mechanism from the tail-motor interaction within KHC. Kinesin cargo-adaptor proteins that bind KLC activated processive movement of the kinesin tetramer but the landing rate of these activated complexes remained low. The addition of MAP7, which specifically binds to the KHC, strongly enhanced activated motor motility by dramatically increasing the landing rate and processivity of the activated kinesin motors. Our results support a model whereby the activity of the kinesin tetramer is regulated by independent tail- and KLC-based inhibition mechanisms, and that cargo-adaptor binding to the KLC directly releases both of these inhibitions. However, we find that a third component, a non-motor MAP is required for robust activity of the activated motor. Thus, human kinesin activity is regulated by a two-factor mechanism comprised of intramolecular allosteric regulation, as well as intermolecular kinesin-adaptor and kinesin-MAP interactions.

scholarly journals HIV-1 Engages a Dynein-Dynactin-BICD2 Complex for Infection and Transport to the Nucleus

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Stephanie K. Carnes ◽  
Jing Zhou ◽  
Christopher Aiken
Keyword(s):  
Heavy Chain ◽  
Intracellular Transport ◽  
Adaptor Proteins ◽  
Target Cells ◽  
Dynein Heavy Chain ◽  
Cell Extracts ◽  
Microtubule Motor ◽  
Dynactin Complex ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) infection depends on efficient intracytoplasmic transport of the incoming viral core to the target cell nucleus. Evidence suggests that this movement is facilitated by the microtubule motor dynein, a large multiprotein complex that interacts with dynactin and cargo-specific adaptor proteins for retrograde movement via microtubules. Dynein adaptor proteins are necessary for activating dynein movement and for linking specific cargoes to dynein. We hypothesized that HIV-1 engages the dynein motor complex via an adaptor for intracellular transport. Here, we show that small interfering RNA depletion of the dynein heavy chain, components of the dynactin complex, and the dynein adaptor BICD2 reduced cell permissiveness to HIV-1 infection. Cell depletion of dynein heavy chain and BICD2 resulted in impaired HIV-1 DNA accumulation in the nucleus and decreased retrograde movement of the virus. Biochemical studies revealed that dynein components and BICD2 associate with capsid-like assemblies of the HIV-1 CA protein in cell extracts and that purified recombinant BICD2 binds to CA assembliesin vitro. Association of dynein with CA assemblies was reduced upon immunodepletion of BICD2 from cell extracts. We conclude that BICD2 is a capsid-associated dynein adaptor utilized by HIV-1 for transport to the nucleus.IMPORTANCEDuring HIV-1 infection, the virus must travel across the cytoplasm to enter the nucleus. The host cell motor protein complex dynein has been implicated in HIV-1 intracellular transport. We show that expression of the dynein heavy chain, components of the dynein-associated dynactin complex, and the dynein adaptor BICD2 in target cells are important for HIV-1 infection and nuclear entry. BICD2 interacts with the HIV-1 capsidin vitro, suggesting that it functions as a capsid-specific adaptor for HIV-1 intracellular transport. Our work identifies specific host proteins involved in microtubule-dependent HIV-1 intracellular transport and highlights the BICD2-capsid interaction as a potential target for antiviral therapy.

scholarly journals Metal-binding propensity in the mitochondrial dynamin-related protein 1

2022 ◽  
Author(s):  
Krishnendu Roy ◽  
Thomas Pucadyil
Keyword(s):  
Membrane Proteins ◽  
Metal Binding ◽  
Adaptor Proteins ◽  
Related Protein ◽  
Native Tissue ◽  
Membrane Fission ◽  
In Vitro Reconstitution ◽  
Mitochondrial Lipid ◽  
Potential Strategy

Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Unlike that seen with the native mitochondrial lipid cardiolipin, metal-bound chelator lipids recruit Drp1 to the membrane but is rendered functionally inactive in membrane fission. Metal-bound chelator beads also recruit Drp1 and represents a potential strategy to deplete or purify the protein from native tissue lysates.

scholarly journals In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Inda Setyawati ◽  
Weronika K Stanek ◽  
Maria Majsnerowska ◽  
Lotteke J Y M Swier ◽  
Els Pardon ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Energy Coupling ◽  
Coupling Factor ◽  
In Vitro System ◽  
In Vitro Reconstitution ◽  
Reconstituted System ◽  
Kinetics Of

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.

scholarly journals Bidirectional Translocation of Neurofilaments along Microtubules Mediated in Part by Dynein/Dynactin

2000 ◽  
Vol 11 (10) ◽  
pp. 3495-3508 ◽  
Author(s):  
Jagesh V. Shah ◽  
Lisa A. Flanagan ◽  
Paul A. Janmey ◽  
Jean-François Leterrier
Keyword(s):  
Spinal Cord ◽  
Molecular Motors ◽  
Motor Proteins ◽  
Motor Complex ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Cytoskeletal Elements

Neuronal cytoskeletal elements such as neurofilaments, F-actin, and microtubules are actively translocated by an as yet unidentified mechanism. This report describes a novel interaction between neurofilaments and microtubule motor proteins that mediates the translocation of neurofilaments along microtubules in vitro. Native neurofilaments purified from spinal cord are transported along microtubules at rates of 100-1000 nm/s to both plus and minus ends. This motion requires ATP and is partially inhibited by vanadate, consistent with the activity of neurofilament-bound molecular motors. Motility is in part mediated by the dynein/dynactin motor complex and several kinesin-like proteins. This reconstituted motile system suggests how slow net movement of cytoskeletal polymers may be achieved by alternating activities of fast microtubule motors.

scholarly journals The intra-mitochondrial O-GlcNAcylation system acutely regulates OXPHOS capacity and ROS dynamics in the heart

2021 ◽  
Author(s):  
Justine Dontaine ◽  
Asma Bouali ◽  
Frederic Daussin ◽  
Laurent Bultot ◽  
Didier Vertommen ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Regulatory Mechanism ◽  
Mitochondrial Proteins ◽  
Cardiac Mitochondria ◽  
Multiple Organs ◽  
Mitochondrial Ros ◽  
In Vitro Reconstitution

Abstract Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we used an in vitro reconstitution assay to characterize the intra-mitochondrial O- GlcNAc system without potential cytoplasmic confounding effects. We compared the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific O-GlcNAcylation inducer. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 displayed increased O-GlcNAcylation in response to NButGT. This was associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of CI substrates, and a striking reduction of mitochondrial ROS release, which could be related to O- GlcNAcylation of subunits within the NADH dehydrogenase module of CI. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.

scholarly journals Kinase-Inactivated ULK Proteins Inhibit Autophagy via Their Conserved C-Terminal Domains Using an Atg13-Independent Mechanism

2008 ◽  
Vol 29 (1) ◽  
pp. 157-171 ◽  
Author(s):  
Edmond Y. W. Chan ◽  
Andrea Longatti ◽  
Nicole C. McKnight ◽  
Sharon A. Tooze
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Intramolecular Interactions ◽  
Regulatory Function ◽  
Independent Mechanism ◽  
Dominant Negative Activity

ABSTRACT The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical roles during the activation of autophagy. Previous studies have demonstrated that the conserved C-terminal domain (CTD) of ULK1 controls the regulatory function and localization of the protein. Here, we explored the role of kinase activity and intramolecular interactions to further understand ULK function. We demonstrate that the dominant-negative activity of kinase-dead mutants requires a 7-residue motif within the CTD. Our data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation and conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains other distinct regions that direct membrane association and interaction with the putative human homologue of Atg13, which we have here characterized. Atg13 is required for autophagy and Atg9 trafficking during autophagy. However, Atg13 does not bind the 7-residue dominant-negative motif in the CTD of ULK proteins nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting that in mammalian cells, the CTD may interact with additional autophagy proteins.

scholarly journals In vitro reconstitution of Sgk3 activation by phosphatidylinositol-3-phosphate

2021 ◽  
Author(s):  
Daniel Pokorny ◽  
Linda Truebestein ◽  
Kaelin D Fleming ◽  
John E Burke ◽  
Thomas A Leonard
Keyword(s):  
Conformational Changes ◽  
Binding Pocket ◽  
Growth Factor Signaling ◽  
Allosteric Activation ◽  
Inhibitor Development ◽  
Allosteric Inhibitor ◽  
Px Domain ◽  
In Vitro Reconstitution ◽  
Phosphatidylinositol 3

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is activated by the phospholipid phosphatidylinositol-3-phosphate (PI3P) downstream of growth factor signaling and by Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers. Here, we show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation by PI3P. We demonstrate that PI3P binding induces large conformational changes in Sgk3 associated with its activation, and that the PI3P binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstituted Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to defining the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

scholarly journals In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

2022 ◽  
Author(s):  
Robert Fagiewicz ◽  
Corinne Crucifix ◽  
Celia Deville ◽  
Bruno Kieffer ◽  
Yves Nomine ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Complexes ◽  
Cytoplasmic Dynein ◽  
Full Length ◽  
Recombinant Production ◽  
In Vitro Characterization ◽  
Microtubule Motor ◽  
Glial Progenitor Cells ◽  
Development In Vitro

The cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is one of the most prominent examples within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis as well as interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2 and cytoplasmic dynein-1. We also describe pH-dependent conformational changes of BicD2 using cryoEM, template-free structure predictions, and biophysical tools. Our results will help defining the biochemical parameters for the invitro reconstitution of higher order BicD2 protein complexes.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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