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1.
Neuron ; 97(5): 1094-1109.e9, 2018 03 07.
Article in English | MEDLINE | ID: mdl-29429936

ABSTRACT

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.


Subject(s)
Actinin/metabolism , Disks Large Homolog 4 Protein/metabolism , Excitatory Postsynaptic Potentials/physiology , Hippocampus/metabolism , Actinin/chemistry , Actinin/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Disks Large Homolog 4 Protein/chemistry , Disks Large Homolog 4 Protein/genetics , Female , HEK293 Cells , Humans , Male , Protein Structure, Secondary , Rats
2.
Methods Mol Biol ; 1448: 237-48, 2016.
Article in English | MEDLINE | ID: mdl-27317185

ABSTRACT

Exosomes are cell-derived small extracellular membrane vesicles (50-100 nm in diameter) actively secreted by a number of healthy and diseased cell types. Exosomes can mediate cellular, tissue, and organ level micro communication under normal and pathological conditions by shuttling proteins, mRNA, and microRNAs. Prior to vesicle molecular profiling, these exosomes can be isolated from conditioned cell media or bodily fluids such as urine and plasma in order to explore the contents and functional relevance. Exosome purification and analyses are a fast-growing research field. Regardless of several advances in exosome purification and analyses methods, research still faces several challenges. Despite tremendous interest in the role of extracellular vesicles, there is no general agreement on dependable isolation protocols. Therefore, there is an urgent need to establish reliable protocol of exosome purification and analysis. Here, we report a simple cost-effective isolation and analysis of cardiac myocyte exosomes from conditioned media.


Subject(s)
Biological Transport/genetics , Exosomes/chemistry , Exosomes/metabolism , Myocytes, Cardiac/metabolism , Acetylcholinesterase/chemistry , Acetylcholinesterase/genetics , Animals , Exosomes/genetics , Humans , MicroRNAs/genetics , Myocytes, Cardiac/chemistry , RNA, Messenger/genetics , Rats , Signal Transduction
3.
Cell ; 159(2): 235-7, 2014 Oct 09.
Article in English | MEDLINE | ID: mdl-25303520

ABSTRACT

Neuronal plasticity depends on plasma membrane Ca(2+) influx, resulting in activity-dependent gene transcription. Calmodulin (CaM) activated by Ca(2+) initiates the nuclear events, but how CaM makes its way to the nucleus has remained elusive. Ma et al. now show that CaMKIIγ transports CaM from cell surface Ca(2+) channels to the nucleus.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Animals
4.
EMBO J ; 33(12): 1341-53, 2014 Jun 17.
Article in English | MEDLINE | ID: mdl-24705785

ABSTRACT

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.


Subject(s)
Calmodulin/metabolism , Hippocampus/cytology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Models, Neurological , Neurons/metabolism , Post-Synaptic Density/metabolism , Animals , Cells, Cultured , Disks Large Homolog 4 Protein , Fluorescence , Histological Techniques , Immunoblotting , Immunoprecipitation , Magnetic Resonance Spectroscopy , Protein Conformation , Rats
5.
J Cardiovasc Pharmacol ; 63(3): 196-206, 2014 Mar.
Article in English | MEDLINE | ID: mdl-23884159

ABSTRACT

The treatment of heart failure (HF) has evolved during the past 30 years with the recognition of neurohormonal activation and the effectiveness of its inhibition in improving the quality of life and survival. Over the past 20 years, there has been a revolution in the investigation of the mitochondrion with the development of new techniques and the finding that mitochondria are connected in networks and undergo constant division (fission) and fusion, even in cardiac myocytes. This has led to new molecular and cellular discoveries in HF, which offer the potential for the development of new molecular-based therapies. Reactive oxygen species are an important cause of mitochondrial and cellular injury in HF, but there are other abnormalities, such as depressed mitochondrial fusion, that may eventually become the targets of at least episodic treatment. The overall need for mitochondrial fission/fusion balance may preclude sustained change in either fission or fusion. In this review, we will discuss the current HF therapy and its impact on the mitochondria. In addition, we will review some of the new drug targets under development. There is potential for effective, novel therapies for HF to arise from new molecular understanding.


Subject(s)
Heart Failure/therapy , Mitochondria, Heart/pathology , Myocytes, Cardiac/pathology , Animals , Drug Design , Heart Failure/physiopathology , Humans , Mitochondria, Heart/metabolism , Mitochondrial Dynamics , Molecular Targeted Therapy , Reactive Oxygen Species/metabolism
6.
Arch Insect Biochem Physiol ; 80(1): 26-41, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22499434

ABSTRACT

Apolipophorin III (apoLp-III) has been known as a lipid transport protein of insects. Recent studies indicated the involvement of apoLp-III in immune reactions and in the control of cell destruction, but no enzymatic activity has so far been detected. In the present study, a protease from the hemolymph of Schistocerca gregaria was purified to homogeneity and its enzymatic activity was examined. Identity as chymotrypsin-like proteinase was established by its high affinity toward bulky aromatic substrates and its catalytic specificity for amide or ester bonds on the synthetic substrates, Suc-Ala-Ala-Pro-Xaa-AMC (where Xaa was Phe, Tyr, Trp, and Lys, and AMC is 7-amino-4-methyl-coumarin) and thiolbenzyl ester substrate Suc-Ala-Ala-Pro-Phe-SBzl. The sensitivity for serine protease and chymotrypsin-specific covalent inhibitors, PMSF, TPCK, and noncovalent inhibitors SGCI, showed that it is a chymotrypsin-like proteinase. It showed its maximum activity at pH 8.0 and 55°C for the hydrolysis of Suc-Ala-Ala-Pro-Tyr-AMC. According to similarities in the amino terminal sequence, molar mass (19 kDa) and retention on reversed-phase analytical high-performance liquid chromatography (HPLC) column, this protein is S. gregaria homologue of Locusta migratoria apoLp-III. Our data suggest that apoLp-III also has an inherent proteolytic activity. Results indicated that S. gregaria apoLp-III is a good catalyst and could be used as a biotechnological tool in food processing and in agricultural biotechnology.


Subject(s)
Apolipoproteins/metabolism , Grasshoppers/enzymology , Hemolymph/enzymology , Insect Proteins/metabolism , Animals , Apolipoproteins/isolation & purification , Insect Proteins/isolation & purification , Serine Proteases/metabolism
7.
Appl Biochem Biotechnol ; 165(7-8): 1779-88, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21976149

ABSTRACT

Apolipophorin III (apoLp-III) from insects and apolipoprotein A-I from humans, are major component of the lipoprotein and share various properties. ApoLp-III is an abundant hemolymph protein. Besides its crucial role in lipid transport, apoLp-III is able to associate with fungal and bacterial membranes and stimulate cellular immune responses. ApoLp-III was isolated and purified from the hemolymph of desert locust Schistocerca gregaria by ion-exchange and reversed-phase chromatography. The purity and the molecular weight of apoLp-III were determined at ∼19,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to similarities in the amino terminal sequence, molar mass and retention on reversed-phase analytical HPLC column, this protein is a Schistocerca gregaria homologue of Locusta migratoria apoLp-III.


Subject(s)
Apolipoproteins/metabolism , Grasshoppers/enzymology , Insect Proteins/metabolism , Amino Acid Sequence , Animals , Apolipoproteins/chemistry , Apolipoproteins/genetics , Apolipoproteins/isolation & purification , Desert Climate , Grasshoppers/chemistry , Grasshoppers/genetics , Hemolymph/chemistry , Hemolymph/enzymology , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/isolation & purification , Molecular Sequence Data , Molecular Weight
8.
EMBO J ; 29(2): 482-95, 2010 Jan 20.
Article in English | MEDLINE | ID: mdl-19942860

ABSTRACT

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the beta(2)-adrenergic receptor (beta(2)AR), the trimeric G(s) protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the beta(2)AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the beta(2)AR is phosphorylated by PKA on beta(2)AR stimulation. Peptides that interfere with the beta(2)AR-GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic beta(2)AR-cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.


Subject(s)
Adenylyl Cyclases/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Receptors, AMPA/analysis , Receptors, AMPA/metabolism , Receptors, Adrenergic, beta-2/metabolism , Adenylyl Cyclases/analysis , Animals , Calcium Channels/metabolism , Cells, Cultured , Cerebral Cortex/metabolism , Cyclic AMP-Dependent Protein Kinases/analysis , Disks Large Homolog 4 Protein , Electrophysiology , GTP-Binding Protein alpha Subunits, Gs/analysis , GTP-Binding Protein alpha Subunits, Gs/metabolism , Gene Expression Regulation , Hippocampus/cytology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neurons/cytology , Rats , Rats, Sprague-Dawley , Receptors, AMPA/genetics , Receptors, Adrenergic, beta-2/analysis
9.
Article in English | MEDLINE | ID: mdl-16820679

ABSTRACT

The structure of the human macrophage inflammatory protein-3alpha (MIP-3alpha) has been determined at 1.81 angstroms resolution by X-ray crystallography. The dimer crystallized in the tetragonal space group I4, with unit-cell parameters a = b = 83.99, c = 57.20 angstroms. The crystals exhibit two molecules in the asymmetric unit. The structure was solved by the molecular-replacement method and the model was refined to a conventional R value of 20.6% (R(free) = 25.7%). MIP-3alpha possesses the same monomeric structure as previously described for other chemokines. However, in addition to limited structural changes in the beta1-beta2 hairpin of monomer B, the electron density is fully defined for a few extra residues at the N- and C-termini of monomer A and the C-terminus of monomer B compared with MIP-3alpha in space group P6(1). As the N-terminal and loop regions have been shown to be critical for receptor binding and signaling, this additional structural information may help in determining the basis of the CCR6 selectivity of MIP-3alpha.


Subject(s)
Chemokines, CC/chemistry , Macrophage Inflammatory Proteins/chemistry , Macrophages/physiology , Binding Sites , Chemokine CCL20 , Chemokines, CC/metabolism , Crystallography, X-Ray , Humans , Macrophage Inflammatory Proteins/metabolism , Receptors, CCR6 , Receptors, Chemokine/chemistry , Receptors, Chemokine/metabolism
10.
J Bacteriol ; 187(21): 7222-31, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16237006

ABSTRACT

Rieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus become important. We report here the structure of free and substrate (indole)-bound forms of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The structure of the Rhodococcus enzyme reveals that, despite a approximately 30% sequence identity between these naphthalene dioxygenases, their overall structures superpose very well with a root mean square deviation of less than 1.6 A. The differences in the active site of the two enzymes are pronounced near the entrance; however, indole binds to the Rhodococcus enzyme in the same orientation as in the Pseudomonas enzyme. Circular dichroism spectroscopy experiments show that the Rhodococcus enzyme has higher thermostability than the naphthalene dioxygenase from Pseudomonas species. The Pseudomonas enzyme has an apparent melting temperature of 55 degrees C while the Rhodococcus enzyme does not completely unfold even at 95 degrees C. Both enzymes, however, show similar unfolding behavior in urea, and the Rhodococcus enzyme is only slightly more tolerant to unfolding by guanidine hydrochloride. Structure analysis suggests that the higher thermostability of the Rhodococcus enzyme may be attributed to a larger buried surface area and extra salt bridge networks between the alpha and beta subunits in the Rhodococcus enzyme.


Subject(s)
Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Oxygenases/chemistry , Oxygenases/metabolism , Rhodococcus/enzymology , Amino Acid Sequence , Binding Sites , Circular Dichroism , Dioxygenases , Enzyme Stability , Models, Molecular , Molecular Sequence Data , Multienzyme Complexes/genetics , Oxygenases/genetics , Protein Conformation , Pseudomonas/enzymology , Sequence Homology, Amino Acid , Temperature , Transition Temperature
11.
J Immunol ; 170(6): 2811-5, 2003 Mar 15.
Article in English | MEDLINE | ID: mdl-12626530

ABSTRACT

One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca(2+) that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca(2+)-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.


Subject(s)
Calcium Signaling/immunology , Lysophospholipids , Macrophages/enzymology , Macrophages/immunology , Mycobacterium tuberculosis/immunology , Phagosomes/immunology , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Sphingosine/analogs & derivatives , Animals , CHO Cells , Calcium/metabolism , Cell Fractionation , Cricetinae , Enzyme Activation , Humans , Macrophage-1 Antigen/physiology , Macrophages/metabolism , Macrophages/microbiology , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/pathogenicity , Phagocytosis/immunology , Phagosomes/enzymology , Phosphotransferases (Alcohol Group Acceptor)/physiology , Sphingosine/biosynthesis , Sphingosine/metabolism , Tuberculosis Vaccines/pharmacology , Vaccines, Inactivated/pharmacology
12.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 12): 2173-4, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12454491

ABSTRACT

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear non-heme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 A, alpha = beta = gamma = 90 degrees, and diffract to 2.3 A resolution.


Subject(s)
Multienzyme Complexes/chemistry , Oxygenases/chemistry , Rhodococcus/enzymology , Crystallization , Crystallography, X-Ray , Dioxygenases , Protein Conformation
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