Involvement of the betaTrCP in the ubiquitination and stability of the HIV-1 Vpu protein

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and targets it to the proteasome for degradation. This process requires the recruitment of human betaTrCP, a component of the Skp1-Cullin-F box (SCF) ubiquitin ligase complex, that interacts with phosphorylated Vpu molecules. Vpu, unlike other ligands of betaTrCP, has never been reported to be degraded. We provide evidence that Vpu, itself, is ubiquitinated and targeted for degradation by the proteasome. We demonstrate that the mutant Vpu2.6, which cannot interact with betaTrCP, is stable and, unlike wild-type Vpu, is not polyubiquitinated. These results suggest that betaTrCP is involved in Vpu polyubiquitination.     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

Structural basis of the properties of an industrially relevant thermophilic xylanase

A thermophilic xylanase from Bacillusstrain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60°C and relatively stable at high temperatures, with a temperature optimum of 75°C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of BacillusD3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or “sticky patches” between pairs of molecules. These “sticky patches” on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase. Proteins 29:77–86, 1997. © 1997 Wiley-Liss, Inc.     

Did the Quaternary climatic fluctuations really influence the tempo and mode of diversification in European rodents?

The objective of this study was to establish whether the Quaternary climatic fluctuations influenced the tempo and mode of diversification in European rodents. Our case study is the subgenus Microtus (Terricola) distributed from western Europe to the Caucasus. Mitochondrial cytochrome b gene sequences from several representatives of all the species were used to generate maximum‐likelihood and Bayesian phylogenetic trees, to estimate divergence times, to identify biogeographic ancestral areas and to study the rate of diversification. Results showed that phylogenetic tree topologies were similar to previous published studies but with a better resolution at some nodes. The origin of Microtus (Terricola) is dated back to approximately 4.05 Myr in the Early Pliocene, and molecular dating for most Terricola species corresponds to several glacial periods of the Pleistocene. Results of the biogeographic ancestral area reconstruction suggest that Microtus (Terricola) diversified from the Caucasus/Turkey/Iran area through western Europe. Several periods of diversity variation were highlighted as follows: two period of diversity increase, between 3 and 2 Myr, and after 1 Myr; two periods of diversity decrease, before 3 Myr, and between 2 and 1 Myr. The diversification rate of Microtus (Terricola) was 0.353 ± 0.004 event/Myr, a rate similar to that of the Muridae family. To conclude, although the Pleistocene glacial conditions had an impact on the speciation events, the Quaternary does not appear however as a period with an exceptional rate of diversification for European rodents.     

Histone H4K20 tri‐methylation at late‐firing origins ensures timely heterochromatin replication

Among other targets, the protein lysine methyltransferase PR‐Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4‐20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S‐phase progression and protects from DNA re‐replication induced by stabilization of PR‐Set7. Using Epstein–Barr virus‐derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4‐20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2‐7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4‐20h‐mediated H4K20 tri‐methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1‐associated origins, which ensure proper replication timing of late‐replicating heterochromatin domains. Altogether, these results reveal Suv4‐20h‐mediated H4K20 tri‐methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.     

Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid

Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses.     

Targeting telomeres to enforce cancer cells to senesce

The telomeres protect the end of chromosomes from being recognized and processed as an accidental double stranded break. In human somatic cells, telomeres shorten progressively with every round of DNA replication, leading to dysfunctional telomeres that trigger cellular senescence or apoptosis depending on the cell type. This telomere erosion appears to play a role in cell renewal, ageing and cancer. Two recent studies demonstrated in mouse that eroded telomeres in cancer cells blocked for apoptosis limit cancer formation by triggering senescence. These results suggest that provoking senescence may provide a way to cure cancer and point to new therapeutical strategies targeting specific telomeric functions. Nevertheless, an important question remains unanswered: does replicative senescence limit tumor formation in human?     

Carbonyl scavenger and antiatherogenic effects of hydrazine derivatives

Reactive carbonyl compounds (RCC) generated by polyunsaturated fatty acid oxidation alter progressively cellular and tissular proteins by forming adducts on free amino groups and thiol residues (carbonyl stress). Carbonyl scavengers may neutralize RCC, but their protective effect in atherosclerosis has not been extensively studied. We report the carbonyl scavenger and antiatherogenic properties of hydrazine derivatives, namely hydralazine, an antihypertensive drug, isoniazid, an antituberculosis agent, and two antidepressants, phenelzine and iproniazid. These drugs were poorly efficient in preventing the oxidation of LDL mediated by smooth muscle cells (SMCs), but inhibited the toxicity of UV-oxidized LDL (oxLDL) and of 4-hydroxynonenal (4-HNE). Hydrazine derivatives prevented the formation of foam cells resulting from LDL oxidation in human macrophagic U937 cells, and blocked the carbonyl stress in SMCs, by inhibiting the decrease in free amino group content, the increase in carbonylated proteins, and the formation of 4-HNE adducts on PDGFR. Experimental studies carried out on apoE-/- mice supplemented with drugs (30 mg/L in drinking water) showed a significant carbonyl stress inhibition correlated with a net reduction of atherosclerotic lesion development. In conclusion, these data indicate that hydrazine derivatives exhibit carbonyl scavenger and antiatherogenic properties, which opens novel therapeutical approaches for atherosclerosis and its cardiovascular complications.