Mitochondrial Membrane Potential in Human Neutrophils Is Maintained by Complex III Activity in the Absence of Supercomplex Organisation

textabstractBackground: Neutrophils depend mainly on glycolysis for their enegry provision. Their mitochondria maintain a membrace potential (ΔΨm), which is usually generated by the repiratory chain complexes. We investigated the source of ΔΨm in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this ΔΨm for the generation of ATP. Methods and Principal Findings: Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but ΔΨm was still decreased byinhibition of complex III, confirming the role of the respiratory chain in maintaining ΔΨm. Complex V did not maintain ΔΨm by consumption of ATP, as has previously been suggested for eosinophils shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were ladding in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercimplex organisation while gaining increased aerobic glycolysis, just like neutrophils. Conclusions: We show that neutrophils can maintain ΔΨm via the glycerol-3-phosphate shuttle, wereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors

Caspase Cleavage Sites in the Human Proteome: CaspDB, a Database of Predicted Substrates

<div><p>Caspases are enzymes belonging to a conserved family of <b><u>c</u>ysteine-dependent <u>asp</u>artic-specific prote<u>ases</u></b> that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The <i>in silico</i> predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency.</p></div

Comparision of CaspDB and Cascleave 2.0 scores.

<p>(A) Probability score comparison of caspase-1 cleavage sites. (B) Caspase-8 cleavage sites. CaspDB and Cascleave 2.0 scores are marked in red and black, respectively.</p

Quality measures of trained classifiers and comparison with publicly available prediction model [44].

<p>Abbreviations: TP – number of true positives, FN-false negatives, FP-false positives, TN-true negatives, ACC-accuracy, PRC-precision, SPC-specificity, MCC-Matthews correlation coefficient, Kappa-Kappa statistical value, RF-Random Forest method, NB- Naïve Bayes, J48-decision tree algorithm, SMO-Sequential Minimal Optimization.</p><p>Quality measures of trained classifiers and comparison with publicly available prediction model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110539#pone.0110539-Piippo1" target="_blank">[44]</a>.</p

Optimized parameter values for trained classifier.

<p>Optimized parameter values for trained classifier.</p

Changes in gene expression of granulocytes during in vivo granulocyte colony-stimulating factor/dexamethasone mobilization for transfusion purposes

The treatment of healthy donors with granulocyte colony-stimulating factor (G-CSF) and dexamethasone results in sufficient numbers of circulating granulocytes to prepare granulocyte concentrates for clinical purposes. Granulocytes obtained in this way demonstrate relatively normal functional behavior combined with a prolonged life span. To study the influence of mobilizing agents on granulocytes, we used oligonucleotide microarrays to identify genes that are differentially expressed in mobilized granulocytes compared with control granulocytes. More than 1000 genes displayed a differential expression pattern, with at least a 3-fold difference. Among these, a large number of genes was induced that encode proteins involved in inflammation and the immune response, such as C-type lectins and leukocyte immunoglobulin-like receptors. Because mobilized granulocytes have a prolonged life span, we focused on genes involved in the regulation of apoptosis. One of the most prominent among these was CAST, the gene encoding calpastatin. Calpastatins are the endogenous inhibitors of calpains, a family of calcium-dependent cysteine proteases recently shown to be involved in neutrophil apoptosis. Transcriptional activity of the CAST gene was induced by G-CSF/dexamethasone treatment both in vivo and in vitro, whereas the protein expression of CAST was stabilized during culture. These studies provide new insight in the genotypic changes as well as in the regulation of the immunologic functions and viability of mobilized granulocytes used for clinical transfusion purposes

ATP levels in neutrophils and PBMC after treatment with respiratory chain inhibitors.

<p>A–F) Neutrophils (▪,□) and PBMC (•,○) were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the mitochondrial uncoupler CCCP (A), or inhibitors for the OXPHOS complexes I-V(F₁), rotenone (B), 3-nitropropionate (3NP; C), antimycin A (D), KCN (E), or aurovertin B (F), repectively. ATP levels were determined with a luciferase-based assay. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p

Lactate levels produced by neutrophils and PBMC after treatment with respiratory chain inhibitors.

<p>A–F) Neutrophils (▪,□) and PBMC (•,○) were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the mitochondrial uncoupler CCCP (A), or inhibitors for the OXPHOS complexes I-V(F₁), rotenone (B), 3-nitropropionate (3NP; C), antimycin A (D), KCN (E), or aurovertin B (F), respectively. Lactate levels were determined with an enzymatic assay. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p