Adaptor protein controlled oligomerization activates the AAA+ protein ClpC

The AAA+ protein ClpC is not only involved in the removal of misfolded and aggregated proteins but also controls, through regulated proteolysis, key steps of several developmental processes in the Gram-positive bacterium Bacillus subtilis. In contrast to other AAA+ proteins, ClpC is unable to mediate these processes without an adaptor protein like MecA. Here, we demonstrate that the general activation of ClpC is based upon the ability of MecA to participate in the assembly of an active and substrate-recognizing higher oligomer consisting of ClpC and the adaptor protein, which is a prerequisite for all activities of this AAA+ protein. Using hybrid proteins of ClpA and ClpC, we identified the N-terminal and the Linker domain of the first AAA+ domain of ClpC as the essential MecA interaction sites. This new adaptor-mediated mechanism adds another layer of control to the regulation of the biological activity of AAA+ proteins.     

Diversity decreases invasion via both sampling and complementarity effects

Complementarity and sampling effects may both contribute to increased invasion resistance at higher diversity. We measured plant invader biomass across a long-term experimental plant diversity gradient. Invader species' biomass was inhibited in more diverse plots, largely because of the presence of strongly competitive C₄ bunchgrasses, consistent with a sampling effect. Invader biomass was negatively correlated with resident root biomass, and positively correlated with soil nitrate concentrations, suggesting that competition for nitrogen limited invader success. Resident root biomass increased and soil nitrate concentrations decreased with the presence of C₄ grasses and also across the diversity gradient, suggesting that diverse plots are more competitive because of the presence of C₄ grasses. In addition to this evidence for a sampling effect, we also found evidence for a complementarity effect. Specifically, the percentage of plots that had lower invader biomass than did the best resident monoculture (i.e. that had invader 'underyielding') increased across the species richness gradient. This pattern cannot be explained by a sampling effect and is a unique signature of complementarity effects. Our results demonstrate the importance of multiple mechanisms by which diversity can increase invasion resistance.     

Protein oxidation and degradation during aging: role in skin aging and neurodegeneration

During aging, the products of oxidative processes accumulate and might disturb cellular metabolism. Among them are oxidized proteins and protein aggregates. On the other hand, in a functioning metabolic system oxidized proteins are degraded, mainly by the proteasome. During aging, however, proteasome activity declines. Therefore, the ability to degrade oxidized proteins is attenuated. The following review summarises the accumulation of oxidized proteins and the decline of the proteasomal system during skin and brain aging including some age-related neurodegenerative processes. The role of protein aggregates will be discussed as a potential reason for the accelerated dysfunction of tissue during aging.     

Isoquinoline-pyridine-based protein kinase B/Akt antagonists: SAR and in vivo antitumor activity

The structure-activity relationships of a series of isoquinoline-pyridine-based protein kinase B/Akt antagonists have been investigated in an effort to improve the major short-comings of the lead compound 3, including poor pharmacokinetic profiles in several species (e.g., mouse i.v. t(1/2) = 0.3 h, p.o. F = 0%). Chlorination at C-1 position of the isoquinoline improved its pharmacokinetic property in mice (i.v. t(1/2) = 5.0 h, p.o. F = 51%) but resulted in >500-fold drop in potency. In a mouse MiaPaCa-2 xenograft model, an amino analog 10y significantly slowed the tumor growth, however was accompanied by toxicity.     

Proteome analysis of mitochondrial outer membrane from Neurospora crassa

The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of LC-MS/MS of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS PMF. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.     

Effects of oral vitamin C supplementation in hemodialysis patients: a proteomic assessment

Evidence indicates that oxidative stress is present in dialysis patients, and is associated with vitamin C deficiency. Limited data are available regarding the effects of vitamin C supplementation on oxidative stress and inflammation markers in these patients. Moreover, there are no data available on plasma polypeptide fingerprints by proteome analysis before and after vitamin C supplementation. Therefore, we analyzed plasma samples from a prospective, randomized, open-labeled trial to assess the effects of oral vitamin C supplementation (250 mg three times per week), to define the plasma polypeptide pattern in hemodialysis patients. Our results reveal that more than 30 polypeptides show significant changes in the dialysis patients in comparison to controls with normal renal function, and that several polypeptides are affected/normalized by oral vitamin C supplementation. These results underline the remarkable potential for proteomics to recognize specific peptide profiles in different pathological situations, which might not be detected by classical methods.     

Degradation of glycated bovine serum albumin in microglial cells

Glycated protein products are formed upon binding of sugars to lysine and arginine residues and have been shown to accumulate during aging and in pathologies such as Alzheimer disease and diabetes. Often these glycated proteins are transformed into advanced glycation end products (AGEs) by a series of intramolecular rearrangements. In the study presented here we tested the ability of microglial cells to degrade BSA-AGE formed by glycation reactions of bovine serum albumin (BSA) with glucose and fructose. Microglial cells are able to degrade BSA-AGEs to a certain degree by proteasomal and lysosomal pathways. However, the proteasome and lysosomal proteases are severely inhibited by cross-linked BSA-AGEs. BSA-AGEs are furthermore able to activate microglial cells. This activation is accompanied by an enhanced degradation of BSA-AGE. Therefore, we conclude that microglial cells are able to degrade glycated proteins, although cross-linked protein-AGEs have an inhibitory effect on proteolytic systems in microglial cells.     

Tocopherol-mediated modulation of age-related changes in microglial cells: turnover of extracellular oxidized protein material

Proteins accumulate during aging and form insoluble protein aggregates. Microglia are responsible for their removal from the brain. During aging, changes within the microglia might play a crucial role in the malfunctioning of these cells. Therefore, we isolated primary microglial cells from adult rats and compared their activation status and their ability to degrade proteins to that of microglial cells isolated from newborn animals. The ability of adult microglial cells to degrade proteins is substantially decreased. However, the preincubation of microglial cells with vitamin E improves significantly the degradation of such modified proteins. The degradation of proteins from apoptotic vesicles is decreased in microglia isolated from adult rats. This might be the result of a suppression of the CD36 receptor due to vitamin E treatment. We concluded that microglial cells isolated from adult organisms have different metabolic properties and seem to be a more valuable model to study age-related diseases.     

Limited degradation of oxidized calmodulin by proteasome: formation of peptides

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H₂O₂ concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.