MAPKAP kinase 2-deficiency prevents neurons from cell death by reducing neuroinflammation--relevance in a mouse model of Parkinson's disease

The inflammatory response in the brain is closely associated with the pathogenesis of degenerative neurological disorders. A role for the p38 stress-activated protein kinase/MAPK-activated protein kinase 2 (MK2) axis in inflammation and apoptosis is well documented. Here, we provide evidence that neurodegeneration can be prevented by eliminating MK2. In primary mesencephalic neuron-glia co-cultures dopaminergic neurons from MK2-deficient (MK2−/−) mice were significantly more resistant to lipopolysaccharide-induced neurotoxicity compared with cells from wild-type mice. This neuroprotection in MK2-deficient cultures was associated with a reduced inflammatory response, especially with reduced production of the inflammatory mediators tumor necrosis factor alpha, keratinocyte-derived chemokine, interleukin-6, and nitric oxide (NO). Interestingly, in primary neuron-enriched cell cultures p38 MAPK, but not MK2, also participates in NO-mediated neuronal cell death. In the MPTP mouse model for Parkinson's disease, MK2-deficient mice show a reduced neuroinflammation and less degeneration of dopaminergic neurons in the substantia nigra after MPTP lesion compared with wild-type mice. In conclusion, our results reveal that MK2 does not directly participate in neuronal cell death, but indirectly contributes to neurodegeneration by the production of neurotoxic substances, such as NO or tumor necrosis factor alpha, from activated glia cells.     

The IL-8 protease SpyCEP/ScpC of group A Streptococcus promotes resistance to neutrophil killing

Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.     

Protein abundance of AKT and ERK pathway components governs cell type‐specific regulation of proliferation

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.     

Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.     

Higher plasma levels of F2-isoprostanes are associated with slower psychomotor speed in healthy older adults

Oxidative stress has been identified as a process which is detrimental to brain health, and associated with age-related cognitive declines. Few studies to-date have examined the relationship between in vivo oxidative stress biomarkers and cognitive performance within healthy elderly populations. The current study investigated the relationship between reaction time and oxidative stress, as measured by blood plasma concentrations of F₂-isoprostanes using a sample of 251 healthy, non-demented, elderly volunteers (Male; 111: Female 140) aged 60–75 years from the Australian Research Council Longevity Intervention (ARCLI) study cohort. A Jensen Box was used in conjunction with the Hick paradigm in order to differentiate simple from choice reaction time (two, four and eight-choice conditions) as well as movement (MT) and decision times (DT). MT, but not DT, was found to be significantly slower for participants in the high F₂-isoprostane group compared to the low F₂-isoprostane group, across all stimulus choices. F₂-isoprostanes, age and Wechsler Abbreviated Scale of Intelligence (WASI) full scale intelligence quotient (IQ) were found to be significant predictors of average MT in the sample as a whole. These findings provide preliminary evidence to suggest that higher levels of oxidative stress may be associated with impaired psychomotor speed in the healthy elderly population.     

Methyl group reorientation under ligand binding probed by pseudocontact shifts

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1–3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.