Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.     

The catalytically inactive precursor of cathepsin D induces apoptosis in human fibroblasts and HeLa cells

In several reports cathepsin D has been implicated in apoptosis. In some systems the effects of agents considered to be mediated by cathepsin D were inhibited in the presence of pepstatin A, an inhibitor of the enzyme. In other studies the effect of a mutant cathepsin D deprived of activity was indistinguishable from that of the normal enzyme. Here we show that in human fibroblasts and in HeLa cells apoptosis can be induced by microinjecting into cytosol either mature cathepsin D or its inactive precursor procathepsin D. The microinjected precursor remains in the uncleaved form. These results confirm that the proapoptotic effect of cathepsin D in the cytosol is independent of its catalytic activity and suggest that the interaction of cathepsin D with the downstream effector does not involve the active site of the enzyme, since in the proenzyme the active site is masked by the prosequence.     

Towards the high-resolution protein structure prediction. Fast refinement of reduced models with all-atom force field

Background  

Although experimental methods for determining protein structure are providing high resolution structures, they cannot keep the pace at which amino acid sequences are resolved on the scale of entire genomes. For a considerable fraction of proteins whose structures will not be determined experimentally, computational methods can provide valuable information. The value of structural models in biological research depends critically on their quality. Development of high-accuracy computational methods that reliably generate near-experimental quality structural models is an important, unsolved problem in the protein structure modeling.     

Large carbon release legacy from bark beetle outbreaks across Western United States

Warmer conditions over the past two decades have contributed to rapid expansion of bark beetle outbreaks killing millions of trees over a large fraction of western United States (US) forests. These outbreaks reduce plant productivity by killing trees and transfer carbon from live to dead pools where carbon is slowly emitted to the atmosphere via heterotrophic respiration which subsequently feeds back to climate change. Recent studies have begun to examine the local impacts of bark beetle outbreaks in individual stands, but the full regional carbon consequences remain undocumented for the western US. In this study, we quantify the regional carbon impacts of the bark beetle outbreaks taking place in western US forests. The work relies on a combination of postdisturbance forest regrowth trajectories derived from forest inventory data and a process‐based carbon cycle model tracking decomposition, as well as aerial detection survey (ADS) data documenting the regional extent and severity of recent outbreaks. We find that biomass killed by bark beetle attacks across beetle‐affected areas in western US forests from 2000 to 2009 ranges from 5 to 15 Tg C yr^?1 and caused a reduction of net ecosystem productivity (NEP) of about 6.1–9.3 Tg C y^?1 by 2009. Uncertainties result largely from a lack of detailed surveys of the extent and severity of outbreaks, calling out a need for improved characterization across western US forests. The carbon flux legacy of 2000–2009 outbreaks will continue decades into the future (e.g., 2040–2060) as committed emissions from heterotrophic respiration of beetle‐killed biomass are balanced by forest regrowth and accumulation.     

Long-wavelength absorbing and fluorescent chameleon labels for proteins, peptides, and amines

Long-wavelength absorbing labels that change their color and fluorescence upon conjugation to proteins and other biomolecules provide two critical advantages over the wealth of conventional amine-reactive labels. At first, the progress of the labeling reaction can be monitored continuously either visually or by spectrometry without prior purification. Then, the labeled biomolecule can be investigated with red or near-infrared light, which minimizes background interference in biological samples. These unique characteristics are met by a group of long-wavelength absorbing cyanine dyes carrying a reactive chloro substituent for nucleophilic substitution with primary amines, which is accompanied by a color change from green to blue. In addition to this so-called chameleon effect, the dyes display an increase in fluorescence during the labeling reaction. Despite their structural similarity, the reactivity of the dyes differs strongly. The fastest labeling kinetics is observed with dye S 0378 as its five-membered ring affords a stabilizing effect on the intermediate carbocation during an S(N)1-type of nucleophilic substitution. The reaction mechanism of the amine-reactive cyanine dyes provides a blueprint for the design of future long-wavelength absorbing chameleon dyes.     

Extracellular DNA is abundant and important for microcolony strength in mixed microbial biofilms

A new approach for quantification of extracellular DNA (eDNA) in mixed biofilms at microscale resolution was developed and combined with other staining techniques to assess the origin, abundance and role of eDNA in activated sludge biofilms. Most eDNA was found in close proximity to living cells in microcolonies, suggesting that most of it originated from an active secretion or alternatively, by lysis of a sub-population of cells. When the staining was combined with fluorescence in situ hybridization for identification of the microorganisms, it was found that the eDNA content varied among the different probe-defined species. The highest amount of eDNA was found in and around the microcolonies of denitrifiers belonging to the genera Curvibacter and Thauera, the ammonium-oxidizing Nitrosomonas and the nitrite-oxidizing Nitrospira. Other floc-formers also produced eDNA, although in lower amounts. The total eDNA content in activated sludge varied from 4 to 52 mg per gram volatile suspended solids in different wastewater treatment plants. Very high local concentrations within some microcolonies were found with up to approximately 300 mg of eDNA per g of organic matter. DNase digestion of activated sludge led to general floc disintegration and disruption of the microcolonies with high eDNA content, implying that eDNA was an important structural component in activated sludge biofilms.     

Mitochondrial Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system

The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/Erv1 disulfide relay system, although Ccs1 is, in contrast to typical substrates, a multidomain protein and lacks twin Cx(n)C motifs. We report on the molecular mechanism of the mitochondrial import of Saccharomyces cerevisiae Ccs1 as the first member of a novel class of unconventional substrates of the disulfide relay system. We show that the mitochondrial form of Ccs1 contains a stable disulfide bond between cysteine residues C27 and C64. In the absence of these cysteines, the levels of Ccs1 and Sod1 in mitochondria are strongly reduced. Furthermore, C64 of Ccs1 is required for formation of a Ccs1 disulfide intermediate with Mia40. We conclude that the Mia40/Erv1 disulfide relay system introduces a structural disulfide bond in Ccs1 between the cysteine residues C27 and C64, thereby promoting mitochondrial import of this unconventional substrate. Thus the disulfide relay system is able to form, in addition to double disulfide bonds in twin Cx(n)C motifs, single structural disulfide bonds in complex protein domains.