Assessment of ab initio models of protein complexes by molecular dynamics.

Determining how proteins interact to form stable complexes is of crucial importance, for example in the development of novel therapeutics. Computational methods to determine the thermodynamically stable conformation of complexes from the structure of the binding partners, such as RosettaDock, might potentially emerge to become a promising alternative to traditional structure determination methods. However, while models virtually identical to the correct experimental structure can in some cases be generated, the main difficulty remains to discriminate correct or approximately correct models from decoys. This is due to the ruggedness of the free-energy landscape, the approximations intrinsic in the scoring functions, and the intrinsic flexibility of proteins. Here we show that molecular dynamics simulations performed starting from a number top-scoring models can not only discriminate decoys and identify the correct structure, but may also provide information on an initial map of the free energy landscape that elucidates the binding mechanism

Computational Modeling of Designed Ankyrin Repeat Protein Complexes with their Targets

Recombinant therapeutic proteins are playing an ever-increasing role in the clinic. High-affinity binding candidates can be produced in a high-throughput manner through the process of selection and evolution from large libraries, but the structures of the complexes with target protein can only be determined for a small number of them in a costly, low-throughput manner, typically by x-ray crystallography. Reliable modeling of complexes would greatly help to understand their mode of action and improve them by further engineering, for example, by designing bi-paratopic binders. Designed ankyrin repeat proteins (DARPins) are one such class of antibody mimetics that have proven useful in the clinic, in diagnostics and research. Here we have developed a standardized procedure to model DARPin–target complexes that can be used to predict the structures of unknown complexes. It requires only the sequence of a DARPin and a structure of the unbound target. The procedure includes homology modeling of the DARPin, modeling of the flexible parts of a target, rigid body docking to ensembles of the target and docking with a partially flexible backbone. For a set of diverse DARPin–target complexes tested it generated a single model of the complex that well approximates the native state of the complex. We provide a protocol that can be used in a semi-automated way and with tools that are freely available. The presented concepts should help to accelerate the development of novel bio-therapeutics for scaffolds with similar properties

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

Impaired immune function in Gulf War Illness

<p>Abstract</p> <p>Background</p> <p>Gulf War Illness (GWI) remains a serious health consequence for at least 11,000 veterans of the first Gulf War in the early 1990s. Our understanding of the health consequences that resulted remains inadequate, and this is of great concern with another deployment to the same theater of operations occurring now. Chronic immune cell dysfunction and activation have been demonstrated in patients with GWI, although the literature is not uniform. We exposed GWI patients and matched controls to an exercise challenge to explore differences in immune cell function measured by classic immune assays and gene expression profiling.</p> <p>Methods</p> <p>This pilot study enrolled 9 GWI cases identified from the Department of Veterans Affairs GWI registry, and 11 sedentary control veterans who had not been deployed to the Persian Gulf and were matched to cases by sex, body mass index (BMI) and age. We measured peripheral blood cell numbers, NK cytotoxicity, cytokines and expression levels of 20,000 genes immediately before, immediately after and 4 hours following a standard bicycle ergometer exercise challenge.</p> <p>Results</p> <p>A repeated-measures analysis of variance revealed statistically significant differences for three NK cell subsets and NK cytotoxicity between cases and controls (p < 0.05). Linear regression analysis correlating NK cell numbers to the gene expression profiles showed high correlation of genes associated with NK cell function, serving as a biologic validation of both the <it>in vitro </it>assays and the microarray platform. Intracellular perforin levels in NK and CD8 T-cells trended lower and showed a flatter profile in GWI cases than controls, as did the expression levels of the perforin gene PRF1. Genes distinguishing cases from controls were associated with the glucocorticoid signaling pathway.</p> <p>Conclusion</p> <p>GWI patients demonstrated impaired immune function as demonstrated by decreased NK cytotoxicity and altered gene expression associated with NK cell function. Pro-inflammatory cytokines, T-cell ratios, and dysregulated mediators of the stress response (including salivary cortisol) were also altered in GWI cases compared to control subjects. An interesting and potentially important observation was that the exercise challenge augments these differences, with the most significant effects observed immediately after the stressor, possibly implicating some block in the NK and CD8 T-cells ability to respond to "stress-mediated activation". This has positive implications for the development of laboratory diagnostic tests for this syndrome and provides a paradigm for exploration of the immuno-physiological mechanisms that are operating in GWI, and similar complex syndromes. Our results do not necessarily elucidate the cause of GWI, but they do reveal a role for immune cell dysfunction in sustaining illness.</p

A Systems Biology Approach Identifies Molecular Networks Defining Skeletal Muscle Abnormalities in Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory process of the lung inducing persistent airflow limitation. Extensive systemic effects, such as skeletal muscle dysfunction, often characterize these patients and severely limit life expectancy. Despite considerable research efforts, the molecular basis of muscle degeneration in COPD is still a matter of intense debate. In this study, we have applied a network biology approach to model the relationship between muscle molecular and physiological response to training and systemic inflammatory mediators. Our model shows that failure to co-ordinately activate expression of several tissue remodelling and bioenergetics pathways is a specific landmark of COPD diseased muscles. Our findings also suggest that this phenomenon may be linked to an abnormal expression of a number of histone modifiers, which we discovered correlate with oxygen utilization. These observations raised the interesting possibility that cell hypoxia may be a key factor driving skeletal muscle degeneration in COPD patients

Benchmark thermochemistry of the C_nH_{2n+2} alkane isomers (n=2--8) and performance of DFT and composite ab initio methods for dispersion-driven isomeric equilibria

The thermochemistry of linear and branched alkanes with up to eight carbons has been reexamined by means of W4, W3.2lite and W1h theories. `Quasi-W4' atomization energies have been obtained via isodesmic and hypohomodesmotic reactions. Our best atomization energies at 0 K (in kcal/mol) are: 1220.04 n-butane, 1497.01 n-pentane, 1774.15 n-hexane, 2051.17 n-heptane, 2328.30 n-octane, 1221.73 isobutane, 1498.27 isopentane, 1501.01 neopentane, 1775.22 isohexane, 1774.61 3-methylpentane, 1775.67 diisopropyl, 1777.27 neohexane, 2052.43 isoheptane, 2054.41 neoheptane, 2330.67 isooctane, and 2330.81 hexamethylethane. Our best estimates for $\Delta H^\circ_{f,298K}$ are: -30.00 n-butane, -34.84 n-pentane, -39.84 n-hexane, -44.74 n-heptane, -49.71 n-octane, -32.01 isobutane, -36.49 isopentane, -39.69 neopentane, -41.42 isohexane, -40.72 3-methylpentane, -42.08 diisopropyl, -43.77 neohexane, -46.43 isoheptane, -48.84 neoheptane, -53.29 isooctane, and -53.68 hexamethylethane. These are in excellent agreement (typically better than 1 kJ/mol) with the experimental heats of formation at 298 K obtained from the CCCBDB and/or NIST Chemistry WebBook databases. However, at 0 K a large discrepancy between theory and experiment (1.1 kcal/mol) is observed for only neopentane. This deviation is mainly due to the erroneous heat content function for neopentane used in calculating the 0 K CCCBDB value. The thermochemistry of these systems, especially of the larger alkanes, is an extremely difficult test for density functional methods. A posteriori corrections for dispersion are essential. Particularly for the atomization energies, the B2GP-PLYP and B2K-PLYP double-hybrids, and the PW6B95 hybrid-meta GGA clearly outperform other DFT functionals.Comment: (J. Phys. Chem. A, in press

Gene Expression during the Generation and Activation of Mouse Neutrophils: Implication of Novel Functional and Regulatory Pathways

As part of the Immunological Genome Project (ImmGen), gene expression was determined in unstimulated (circulating) mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes), thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1β, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the ImmGen regulatory model provide a basis for additional hypothesis-based research on the importance of changes in gene expression in neutrophils in different conditions