Dynamin: characteristics,mechanism of action and function

Dynamin - a member of the GTP-ase protein family - is essential for many intracellular membrane trafficking events in multiple endocytic processes. The unique biochemical features of dynamin - especially its propensity to assemble - enable severing the nascent vesicles from the membrane. The mechanism of dynamin's action is still a subject of debate - whether it functions as a mechanochemical enzyme or a regulatory GTPase. The GTPase domain of dynamin contains three GTP-binding motifs. This domain is very conservative across the species, including that recently cloned by us in the unicellular eukaryote Paramecium. Dynamin interacts with a number of partners such as endophilin and proteins involved in coordination of endocytosis with motor molecules. A growing body of evidence indicates that dynamin and dynamin-related proteins are involved both in pathology and protection against human diseases. The most interesting are dynamin-like Mx proteins exhibiting antiviral activity.     

Dual targeting and function of a protease in mitochondria and chloroplasts

Here we show, using the green fluorescent protein (GFP) fusion system, that an Arabidopsis thaliana zinc-metalloprotease (AtZn-MP) is targeted to both mitochondria and chloroplasts. A deletion mutant lacking the amino-terminal 28 residues, with translation initiation at the second methionine residue, was imported into chloroplasts only. However, a mutated form of the full-length targeting peptide, in which the second methionine residue is changed to leucine, was imported to both organelles. No GFP fluorescence was detected when a frame-shift mutation was introduced between the first and second ATG codons of the Zn-MP–GFP construct, suggesting no alternative translational initiation. Our results show that the dual targeting of the Zn-MP is due to an ambiguous targeting peptide. Furthermore, we show that the recombinant AtZn-MP degrades mitochondrial and chloroplastic targeting peptides, indicating its function as a signal peptide degrading protease in both mitochondria and chloroplasts.     

Relationship of demethylation processes to veratric acid concentration and cell density in cultures of Rhodococcus erythropolis

The aim of this study was to investigate the correlation between veratrate degradation, veratric acid concentration and cell density in Rhodococcus erythropolis cultures. The optimum culture conditions for veratrate demethylation proved to be a cell density of A(660)=1 and a concentration of 0.02% veratrate. All the products of demethylation (i.e. vanillic and protocatechuic acids) were found to be present and correlated with the appearance of high levels of free radicals and formaldehyde after contact of the cells with veratrate. Demethylation was accompanied by oscillatory changes in the levels of endogenous oxygen uptake and phenolic products. Changes in veratrate concentration and cell density caused a disturbance in the demethylation process and also in the efficiency of phenolics, formaldehyde and reactive oxygen species.     

Characterization of the targeting signal of dual-targeted pea glutathione reductase

We investigated the dual targeting signal of pea glutathione reductase (GR) that had been previously shown to be capable of targeting the passenger protein phosphinothricin acetyl transferase to mitochondria and chloroplasts in vivo. We confirmed that GR was imported into mitochondria and chloroplasts in vitro. Rupture of the outer mitochondrial membrane after the import assay indicated that GR was imported into both the intermembrane space and the matrix. Changing positive and hydrophobic residues in the targeting signal we investigated if dual targeting of GR was due to an overlapping or separate signal. Overall single mutations had a greater effect on mitochondrial import compared to chloroplasts, especially those on positive residues. Precursors containing both positive and hydrophobic residue mutations (double mutants) indicated that there might be some redundancy in targeting information for chloroplastic import as double mutants had a greater effect than predicted from the single mutants. Fusion of the targeting signal to the green fluorescent protein (GFP) followed by transient transformation indicated that this signal was only capable of targeting this passenger protein to plastids. Additionally, fusion of the complete coding sequence of GR to GFP also resulted in an exclusive chloroplastic localization. Mutations in the targeting signal that reduced import into plastids in vitro also displayed altered patterns of GFP localizations in vivo. These results indicate that some residues in the signal for dual localisation of GR play a role in both mitochondrial and chloroplastic import, and thus the signal is overlapping.     

Dantrolene inhibits NMDA-induced 45Ca uptake in cultured cerebellar granule neurons

Dantrolene is an inhibitor of a skeletal muscle subtype of ryanodine receptors that stabilizes intracellular calcium concentrations and exerts neuroprotective effects in neurons submitted to excitotoxic challenges. The mechanisms of dantrolene-induced neuroprotection are not clear. In this study, using a model of cultured rat cerebellar granule neurons, we demonstrated that dantrolene inhibits NMDA-evoked 45Ca uptake, indicating that this drug may inhibit the activity of NMDA receptor channels. Primary neuronal cultures were incubated for 10 min in Mg(2+)-free ionic medium with NMDA and 45Ca in the presence of different concentrations of dantrolene, then radioactivity in neurons was measured by liquid scintillation spectroscopy. The results demonstrated that dantrolene, applied at micromolar concentrations, inhibits NMDA-evoked 45Ca uptake in neurons in a dose-dependent manner. DMSO, a vehicle to dantrolene, in concentrations used in this study had no effect on NMDA-evoked 45Ca uptake. These results, indicating that dantrolene inhibits activation of the NMDA receptors, might at least partially explain the mechanisms of a dantrolene-evoked protection of neurons against excitotoxicity mediated by agonists of NMDA receptors.     

Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg(2+)-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC(50) of 17.4mM, whereas EC(50) for L-glutamate was 0.17 mM. Addition of 50 microM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 microM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25 mM D,L-homocysteine with 50 microM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 microM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca(2+) to neurones.     

Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium

Molecular search for the homologues of the mammalian proteins in the unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking is discussed. We cloned and sequenced the gene fragments encoding the following components participating in endosome formation, sorting and maturation of the proprotein precursors, respectively, dynamin 2, Rab7 and furin. There is a proof that all these genes are expressed in this unicellular organism. The function of the identified immunoanalogues of the above described components of Paramecium endocytic machinery as well as a high degree of sequence homology to the respective human counterparts points to the evolutionary conservancy of these pathways.     

Enhanced glycerol 3-phosphate dehydrogenase activity in adipose tissue of obese humans

The primary purpose of this investigation was to determine whether adipose tissue glycerol 3-phosphate dehydrogenase activity is associated with human obesity. The data presented in this paper indicate that the glycerol 3-phosphate dehydrogenase activity in adipose tissue from morbidly obese subjects is approximately 2-fold higher than from lean individuals. Moreover, positive correlation between adipose tissue glycerol 3-phosphate dehydrogenase activity and body mass index (BMI) (r = 0.5; p < 0.01) was found. In contrast, the adipose tissue fatty acid synthase (FAS) and ATP-citrate lyase (ACL) activities in morbidly obese patients are significantly lower than in lean subjects. Furthermore, negative correlation between adipose tissue FAS activity and BMI (r = –0.3; p < 0.05) as well as between ACL activity and BMI (r = –0.3; p < 0.05) was found.These data indicate that elevated glycerol 3-phosphate dehydrogenase might contribute to the increase of triacylglycerol (TAG) synthesis in obese subjects, however, fatty acids necessary for glycerol 3-phosphate esterification must be derived (because of lower FAS and ACL activities) mainly from TAG in circulating lipoproteins formed in liver (VLDL), and/or from the intake with food (chylomicrons).The conclusion is, that the enhanced activity of glycerol 3-phosphate dehydrogenase, and hence the generation of more glycerol 3-phosphate in adipose tissue offers a novel explanation for increased TAG production in adipose tissue of obese subjects.     

Non-homologous DNA end joining

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.