Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of guanine nucleotides on the intrinsic and extrinsic fluorescence properties of dynamin were assessed. The intrinsic Trp (tryptophan) fluorescence spectra of purified recombinant dynamin-1 and -2 were very similar, with a maximum at 332 nm. Collisional quenching by KI was weak (approximately 30%), suggesting that the majority of Trp residues are buried. Binding of guanine nucleotides decreased intrinsic fluorescence by 15-20%. Titration of the effects showed that GTP and GDP bound to a single class of non-interacting sites in dynamin tetramers with apparent dissociation constants (K(d)) values of 5.4 and 7.4 microM (dynamin-1) and 13.2 and 7.1 microM (dynamin-2) respectively. Similar dissociation constant values for both nucleotides were obtained by titrating the quenching of IAEDANS [N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine]-labelled dynamin-2. Despite the similar binding affinities, GTP and GDP result in different conformations of the protein, as revealed by sensitivity to proteinase K fragmentation. Dynamins contain five Trp residues, of which four are in the PH domain (pleckstrin homology domain) and one is in the C-terminal PRD (proline/arginine-rich domain). Guanine nucleotides quenched fluorescence emission from a truncated (DeltaPRD) mutant dynamin-1 to the same extent as in the full-length protein, suggesting conformational coupling between the G (groove)-domain and the PH domain. Efficient resonance energy transfer from PH domain Trp residues to bound mant-GTP [where mant stands for 2'-(3')-O-(N-methylanthraniloyl)] suggests that the G-domain and PH domain are in close proximity (5-6 nm). Promotion of dynamin-2 oligomerization, by reduction in ionic strength or increasing protein concentration, had little effect on intrinsic dynamin fluorescence. However, fluorescence emission from IAEDANS.dynamin-2 showed a significant spectral shift on oligomerization. In addition, energy transfer was observed when oligomerization was promoted in mixtures of IAEDANS.dynamin-2 and 4-(4-dimethylaminophenylazo)benzoic acid-coupled dynamin-2, an effect that was counteracted by GTP but not GDP.
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PMID:Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence. 1595 62

ALpha-synuclein (alpha-syn) has been known to be a key player of the pathogenesis of Parkinson's disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of alpha-syn into cells has also been observed. In this study, we observed that dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric alpha-syn into BV-2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of alpha-syn internalization in dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-treated BV-2 cells. Furthermore, inhibition of clathrin-, caveolae-, and dynamin-dependent endocytosis did not prevent the internalization of alpha-syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of alpha-syn. In addition, we further confirmed these observations by co-culture system of BV-2 cells and alpha-syn-over-expressing SH-SY5Y cells. These findings suggest that extracellular alpha-syn is internalized into microglia via GM1 as well as hitherto-unknown protein receptors in clathrin-, caveolae-, and dynamin-independent, but lipid raft-dependent manner. Elucidation of the mechanism involved in internalization of alpha-syn should be greatly helpful in the development of new treatments of alpha-syn-related neurodegenerative diseases.
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PMID:On the mechanism of internalization of alpha-synuclein into microglia: roles of ganglioside GM1 and lipid raft. 1945 4