scholarly journals Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins

1996 ◽  
Vol 319 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Michael E CHEETHAM ◽  
Brian H. ANDERTON ◽  
Antony P. JACKSON
Keyword(s):  
Heat Shock ◽  
Molecular Chaperones ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
General Factor ◽  
Vesicle Preparation ◽  
Specific Proteins ◽  
Kinetics Of ◽  
The Brain

The uncoating of clathrin-coated vesicles can be mediated in vitro by the ‘uncoating ATPase’ that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Athough we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

scholarly journals Kinetics of a nucleoside release from lactide-caprolactone and lactide-glycolide polymers in vitro.

2000 ◽  
Vol 47 (1) ◽  
pp. 59-64
Author(s):  
T Kryczka ◽  
P Grieb ◽  
M Bero ◽  
J Kasperczyk ◽  
P Dobrzynski
Keyword(s):  
Formation Rate ◽  
Local Treatment ◽  
Extracellular Fluid ◽  
Brain Extracellular Fluid ◽  
Artificial Cerebrospinal Fluid ◽  
Fluid Formation ◽  
Further Development ◽  
Kinetics Of ◽  
The Brain

We assessed the rate of release of a model nucleoside (adenosine, 5%, w/w) from nine different lactide-glycolide or lactide-caprolactone polymers. The polymer discs were eluted every second day with an artificial cerebrospinal fluid at the elution rate roughly approximating the brain extracellular fluid formation rate. Adenosine in eluate samples was assayed by HPLC. Three polymers exhibited a relatively constant release of adenosine for over four weeks, resulting in micromolar concentrations of nucleoside in the eluate. This points to the necessity of further development of polymers of this types as intracerebral nucleoside delivery systems for local treatment of brain tumors.

The response of the centrosome to heat shock and related stresses in a Drosophila cell line

1990 ◽  
Vol 96 (3) ◽  
pp. 403-412
Author(s):  
A. Debec ◽  
A.M. Courgeon ◽  
M. Maingourd ◽  
C. Maisonhaute
Keyword(s):  
Heat Shock ◽  
Microtubule Assembly ◽  
Close Link ◽  
Drosophila Cell ◽  
Shock Response ◽  
Kinetics Of ◽  
Drosophila Cell Line ◽  
Good Agreement ◽  
Cell Division Control

The centrosome of Drosophila melanogaster cells cultured in vitro has been followed by immunofluorescence techniques with the Bx63 antibody of Frasch and Saumweber. After a heat shock, the centrosome labelling becomes very small and finally disappears after 30 min. Other heat-shock protein (hsp) inducers such as ethanol, arsenite and ecdysterone lead to the same disappearance. Moreover, the functional ability of centrosomes to nucleate microtubule assembly is inhibited by these treatments, particularly by heat shock, ethanol and ecdysterone. Two other hsp inducers, cadmium chloride and hydrogen peroxide, do not affect the centrosome seriously. With the exception of cadmium, the rapidity and the intensity of hsp induction are in good agreement with the kinetics of alteration of the organelle. We propose that a close link exists between the heat-shock response and the centrosome and that the physiological induction of hsps could be reinterpreted in terms of cell division control.

scholarly journals The Role of Dynamin and Its Binding Partners in Coated Pit Invagination and Scission

2001 ◽  
Vol 152 (2) ◽  
pp. 309-324 ◽  
Author(s):  
Elaine Hill ◽  
Jeroen van der Kaay ◽  
C. Peter Downes ◽  
Elizabeth Smythe
Keyword(s):  
Plasma Membrane ◽  
Sh3 Domain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Binding Partners ◽  
Late Stages

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain–containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

scholarly journals Insulin differentially affects the distribution kinetics of amyloid beta 40 and 42 in plasma and brain

2017 ◽  
Vol 38 (5) ◽  
pp. 904-918 ◽  
Author(s):  
Suresh Kumar Swaminathan ◽  
Kristen M Ahlschwede ◽  
Vidur Sarma ◽  
Geoffry L Curran ◽  
Rajesh S Omtri ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Amyloid Beta Peptides ◽  
Beta Peptides ◽  
Distribution Kinetics ◽  
In Vitro Bbb Model ◽  
First Time ◽  
Kinetics Of ◽  
The Brain ◽  
Distinct Distribution

Impaired brain clearance of amyloid-beta peptides (Aβ) 40 and 42 across the blood–brain barrier (BBB) is believed to be one of the pathways responsible for Alzheimer’s disease (AD) pathogenesis. Hyperinsulinemia prevalent in type II diabetes was shown to damage cerebral vasculature and increase Aβ accumulation in AD brain. However, there is no clarity on how aberrations in peripheral insulin levels affect Aβ accumulation in the brain. This study describes, for the first time, an intricate relation between plasma insulin and Aβ transport at the BBB. Upon peripheral insulin administration in wild-type mice: the plasma clearance of Aβ40 increased, but Aβ42 clearance reduced; the plasma-to-brain influx of Aβ40 increased, and that of Aβ42 reduced; and the clearance of intracerebrally injected Aβ40 decreased, whereas Aβ42 clearance increased. In hCMEC/D3 monolayers (in vitro BBB model) exposed to insulin, the luminal uptake and luminal-to-abluminal permeability of Aβ40 increased and that of Aβ42 reduced; the abluminal-to-luminal permeability of Aβ40 decreased, whereas Aβ42 permeability increased. Moreover, Aβ cellular trafficking machinery was altered. In summary, Aβ40 and Aβ42 demonstrated distinct distribution kinetics in plasma and brain compartments, and insulin differentially modulated their distribution. Cerebrovascular disease and metabolic disorders may disrupt this intricate homeostasis and aggravate AD pathology.

scholarly journals The Unique Chaperone Operon of Thermotoga maritima: Cloning and Initial Characterization of a Functional Hsp70 and Small Heat Shock Protein

1999 ◽  
Vol 181 (14) ◽  
pp. 4237-4244 ◽  
Author(s):  
Edward T. Michelini ◽  
Gregory C. Flynn
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Molecular Chaperones ◽  
Atp Hydrolysis ◽  
Thermotoga Maritima ◽  
Small Heat Shock Protein ◽  
Chaperone Protein ◽  
Operon Organization

ABSTRACT The hyperthermophilic eubacterium Thermotoga maritimapossesses an operon encoding an Hsp70 molecular chaperone protein and a protein with meaningful homology to the small heat shock protein family of chaperones. This represents the first demonstrated co-operon organization for these two important classes of molecular chaperones. We have cloned and initially characterized these proteins as functional chaperones in vitro: the Hsp70 is capable of ATP hydrolysis and substrate binding, and the small heat shock protein can suppress protein aggregation and stably bind a refolding-competent substrate. In addition, the primary sequence of the Hsp70 is used to infer the phylogenetic relationships of T. maritima, one of the deepest-branching eubacteria known.

scholarly journals Brain clathrin and clathrin-associated proteins.

1982 ◽  
Vol 201 (2) ◽  
pp. 297-304 ◽  
Author(s):  
M P Lisanti ◽  
W Schook ◽  
N Moskowitz ◽  
C Ores ◽  
S Puszkin
Keyword(s):  
Cerebral Cortex ◽  
Limited Proteolysis ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Lattice Structures ◽  
Latex Particles ◽  
Associated Proteins ◽  
Sepharose 4B

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.

Purification, crystallization and preliminary X-ray crystallographic studies of S. cerevisiae Hsp40 Sis1

1999 ◽  
Vol 55 (6) ◽  
pp. 1234-1236 ◽  
Author(s):  
Bingdong Sha ◽  
Douglas Cyr
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Atpase Activity ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Heat Shock Protein 70 ◽  
Peptide Binding ◽  
X Ray ◽  
Cellular Processes

Heat-shock protein 70 (Hsp70), one of the major molecular chaperones, has been shown to play a central role in many cellular processes. Heat-shock protein 40 (Hsp40) works as a co-chaperone for Hsp70. Hsp40, bound by unfolded polypeptide, can interact directly with Hsp70 to stimulate the ATPase activity of Hsp70. Hsp40 can also bind to unfolded polypeptides and prevent them from aggregating in vitro, thus acting as an independent molecular chaperone. The S. cerevisiae Hsp40 Sis1 C-terminal peptide-binding domain has been crystallized. The crystals diffract to 2.7 Å and belong to space group P41212 or P43212 with a = 73.63, c = 80.16 Å. The structure determination by the MAD method is under way.

Some properties of aminopeptidase associated with rat brain cortical synaptosomes

1983 ◽  
Vol 61 (11) ◽  
pp. 1185-1190 ◽  
Author(s):  
William W.-C. Chan ◽  
Wolfgang Demmer ◽  
Karl Brand
Keyword(s):  
Rat Brain ◽  
Leucine Aminopeptidase ◽  
Single Species ◽  
Bound State ◽  
Spectrophotometric Assay ◽  
Diethyl Pyrocarbonate ◽  
Kinetics Of ◽  
The Brain

To understand the breakdown of peptides in the brain, we studied the aminopeptidase associated with synaptosome particles. A continuous spectrophotometric assay in stirred cuvettes was used to follow the kinetics of inactivation by EDTA and by diethyl pyrocarbonate. The sensitivity of the enzyme towards puromycin and leucine hydroxamate was also determined. The results are consistent with the presence of a single species of aminopeptidase in freshly prepared synaptosome. This enzyme is capable of degrading Met-enkephalin in vitro and is distinct from microsomal leucine aminopeptidase. Storage of synaptosomes by freezing and subsequent thawing changed some properties of the enzyme and partially solubilized the enzyme. These studies suggest that there are advantages in studying the enzyme in its native particle-bound state.

Heat shock of Drosophila melanogaster induces the synthesis of new messenger RNAs and proteins

1978 ◽  
Vol 283 (997) ◽  
pp. 391-406 ◽  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Messenger Rnas ◽  
Sodium Dodecylsulphate ◽  
Shock Proteins ◽  
Kinetics Of

The heat shock proteins, labelled in vivo with [ 35 S]methionine, were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis and fingerprinted after tryptic digestion. Eight distinct heat shock polypeptides were characterized in this way. Heat shock messenger RNAs were isolated and partially purified. Assayed in vitro for protein synthesis, they were found to code for heat shock polypeptides. Some parameters of the kinetics of in vivo synthesis of the heat shock proteins are presented.

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