Insights into FlaI Functions in Archaeal Motor Assembly and Motility from Structures,Conformations, and Genetics

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Highlights? Cross-subunit interface forms building block for transmission of ATPase force ? Structures and mutants reveal functional components for assembly and motility ? The directionality of movement explains FlaI activities and GspE differences ? The phosphate released by ATP hydrolysis triggers subunit rearrangements     

Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts

The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.     

The role of NADPH oxidase (NOX) enzymes in neurodegenerative disease

Recently, mounting evidence implicating reactive oxygen species (ROS) generated by NADPH oxidase (NOX) enzymes in the pathogenesis of several neurodegenerative diseases including Amyotrophic lateral sclerosis (ALS), Alzheimer’s (AD), Parkinson’s (PD) and polyglutamine disease, have arisen. NOX enzymes are transmembrane proteins and generate reactive oxygen species by transporting electrons across lipid membranes. Under normal healthy conditions, low levels of ROS produced by NOX enzymes have been shown to play a role in neuronal differentiation and synaptic plasticity. However, in chronic neurodegenerative diseases over-activation of NOX in neurons, as well as in astrocytes and microglia, has been linked to pathogenic processes such as oxidative stress, exitotoxicity and neuroinflammation. In this review, we summarize the current knowledge about NOX functions in the healthy central nervous system and especially the role of NOX enzymes in neurodegenerative disease processes.     

Isolation of brain mitochondria from neonatal mice

Mitochondria are key contributors to many forms of cell death including those resulting from neonatal hypoxic-ischemic brain injury. Mice have become increasingly popular in studies of brain injury, but there are few reports evaluating mitochondrial isolation procedures for the neonatal mouse brain. Using evaluation of respiratory activity, marker enzymes, western blotting and electron microscopy, we have compared a previously published procedure for isolating mitochondria from neonatal mouse brain (method A) with procedures adapted from those for adult rats (method B) and neonatal rats (method C). All three procedures use Percoll density gradient centrifugation as a key step in the isolation but differ in many aspects of the fractionation procedure and the solutions used during fractionation. Methods A and B both produced highly enriched fractions of well-coupled mitochondria with high rates of respiratory activity. The fraction from method C exhibited less preservation of respiratory properties and was more contaminated with other subcellular components. Method A offers the advantage of being more rapid and producing larger mitochondrial yields making it useful for routine applications. However, method B produced mitochondria that were less contaminated with synaptosomes and associated cytosolic components that suits studies that have a requirement for higher mitochondrial purification.     

Interactions of the natural product kendomycin and the 20S proteasome

Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds, the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin (KM) was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of KM–proteasome interactions in vitro and in vivo, including complex structures of wild type and mutant yeast 20S proteasome with KM. Our results provide evidence for a polypharmacological mode of action for KM's cytotoxic effect on cancer cells.     

Experiences of Borna disease virus infection in Sweden

In the early 1970s a fatal neurological disorder in cats was reported in the areas around Lake M?laren in central Sweden. The major signs were hind-leg ataxia, as well as absence or marked decrease in postural reactions and in some cases behavioural changes. The pathology of the disorder was characterized as a non-suppurative meningoencephalomyelitis, but the etiology was not determined. Almost twenty years later, the disorder now known as staggering disease (SD), was further characterized both clinically and pathologically. The same histopathological picture was seen as in the previous study, with inflammatory nodules, neuronal degeneration and perivascular cuffs mainly consisting of lymphocytes. The most severe inflammatory changes were seen in the grey matter of the brain stem, basal ganglia and hippocampus. Clinically the same major neurological signs were seen. Although the cats were examined for several known infectious agents causing central nervous system (CNS) disturbances, no etiological cause of SD was determined.     

A non-conservative polymorphism in the IL-6 signal transducer (IL6ST)/gp130 is associated with myocardial infarction in a hypertensive population

Inflammation is a key component in the development of atherosclerosis, and myocardial infarction (MI); therefore we investigated the association between an interleukin-6 signal transducer (IL6ST)/gp130 polymorphism, gp130 function and risk of MI. Structural modeling suggested that a non-conservative single nucleotide polymorphism in the gp130, Gly148Arg, can change the stability and functional properties of the molecule. In vitro studies were done with BAF/3 cells lacking endogenous gp130. Cells stably transfected with the gp130 148Arg variant proliferated less and showed slightly lower STAT-3 phosphorylation in response to gp130 stimulation as compared to cells transfected with gp130 148Gly. In a prospectively followed hypertensive cohort we identified 167 patients who suffered a MI during the study and compared them to matched controls (mean age 57 years, 73% males, n=482). Carriers of the 148Arg variant (f(Arg)=0.12) of the gp130 receptor had decreased odds ratio for MI in univariate analysis (0.56, 95% CI 0.34-0.91, p=0.02). In conclusion, a genetically determined structural variant of the IL-6 receptor subunit gp130 is, independently of other known risk factors, associated with decreased risk of MI. The variant is also associated with decreased IL-6 responsiveness and could lead to a configuration change in the gp130 receptor.