Ab initio and template-based prediction of multi-class distance maps by two-dimensional recursive neural networks

Background

Prediction of protein structures from their sequences is still one of the open grand challenges of computational biology. Some approaches to protein structure prediction, especially ab initio ones, rely to some extent on the prediction of residue contact maps. Residue contact map predictions have been assessed at the CASP competition for several years now. Although it has been shown that exact contact maps generally yield correct three-dimensional structures, this is true only at a relatively low resolution (3–4 Å from the native structure). Another known weakness of contact maps is that they are generally predicted ab initio, that is not exploiting information about potential homologues of known structure.

Results

We introduce a new class of distance restraints for protein structures: multi-class distance maps. We show that C _α trace reconstructions based on 4-class native maps are significantly better than those from residue contact maps. We then build two predictors of 4-class maps based on recursive neural networks: one ab initio, or relying on the sequence and on evolutionary information; one template-based, or in which homology information to known structures is provided as a further input. We show that virtually any level of sequence similarity to structural templates (down to less than 10%) yields more accurate 4-class maps than the ab initio predictor. We show that template-based predictions by recursive neural networks are consistently better than the best template and than a number of combinations of the best available templates. We also extract binary residue contact maps at an 8 Å threshold (as per CASP assessment) from the 4-class predictors and show that the template-based version is also more accurate than the best template and consistently better than the ab initio one, down to very low levels of sequence identity to structural templates. Furthermore, we test both ab-initio and template-based 8 Å predictions on the CASP7 targets using a pre-CASP7 PDB, and find that both predictors are state-of-the-art, with the template-based one far outperforming the best CASP7 systems if templates with sequence identity to the query of 10% or better are available. Although this is not the main focus of this paper we also report on reconstructions of C _α traces based on both ab initio and template-based 4-class map predictions, showing that the latter are generally more accurate even when homology is dubious.

Conclusion

Accurate predictions of multi-class maps may provide valuable constraints for improved ab initio and template-based prediction of protein structures, naturally incorporate multiple templates, and yield state-of-the-art binary maps. Predictions of protein structures and 8 Å contact maps based on the multi-class distance map predictors described in this paper are freely available to academic users at the url http://distill.ucd.ie/.     

The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1): mRNA cloning, structure analysis and comparison with mammalian homologues

Background  

Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX) -producing bacteria.     

Coupling of insulin receptors to glucose transport: a temperature-dependent time lag in activation of glucose transport.

We have studied the ability of occupied insulin receptors to activate (or couple to) the glucose transport system in isolated rat adipocytes. Maximal insulin action is seen when only a small proportion (<10%) of the receptors is occupied, and this fraction can be rapidly filled (<5 s) at an insulin concentration of 100 ng/ml. Additionally, control studies show that when the extracellular glucose concentration is tripled, the rate of transport triples within 10 s, indicating that changes in transport activity can be observed nearly instantaneously. Therefore, when cells are exposed to a high insulin concentration (100 ng/ml), any delay in the onset of insulin action beyond this time must be due to the time required for coupling of occupied insulin receptors to the glucose transport system. At 24 °C there is a lag of at least 200 s after insulin addition before a significant stimulation of 2-deoxyglucose transport is seen. The length of this lag phase is temperature dependent, decreasing to 45 s at 37 °C. An Arrhenius plot of the coupling lag is linear, with an activation energy of 25 kcal/mol. After the delay in the onset of initial transport activation the full response appears in a gradual manner, requiring 20 min at 24 °C to attain maximal stimulation. The time required for the full insulin response to appear is also temperature dependent, decreasing to 5 min at 37 °C. Similar results were obtained for the kinetics of insulin activation of 3-O-methyl glucose transport. Thus, the coupling of insulin receptors to the glucose transport system can be divided into two components: an initial absolute time lag followed by a gradual incremental process before the maximal, or full, effect of insulin is achieved. In conclusion, (1) there is an absolute delay in the onset of the insulin's initial action on glucose transport, (2) after an initial delay, activation of transport proceeds in a gradual manner, and (3) the coupling process between insulin receptors and the glucose transport system is temperature dependent and can be described by a linear Arrhenius plot. This suggests that the rate of activation is not limited by membrane fluidity.     

Internalization and degradation of a low affinity insulin ([LeuB24]insulin) by rat adipocytes

In Halobacterium halobium tactic responses towards light and chemoeffectors are accompanied by changes in the methylation level of methyl-accepting chemotaxis proteins (MCP). Taxis towards green light absorbed by the bacteriorhodopsin proton pump appears to be governed by Δ$\text{μ}$_H⁺-sensing. The addition of CCCP, an uncoupler, prevented the increase of MCP methylation in response to green light illumination, but had no effect on CH₃-incorporation followed by the addition of the attractants glucose, leucine and histidine. Similarly, CCCP did not change MCP demethylation in response to blue light illumination, a repelling stimuli.The sensitivity to an uncoupler of methylation linked to Δ$\text{μ}$_H⁺-mediated green light taxis is to be expected, while the independence of demethylation caused by the blue light of CCCP is an indication that in the latter case a specific photoreceptor governs phototaxis. Informed processing from the blue light receptor to MCP does not involve a change in the membrane potential.     

Properties of a human insulin receptor with a COOH-terminal truncation. I. Insulin binding, autophosphorylation, and endocytosis

In order to test the contribution of the insulin receptor COOH terminus to insulin action, a truncation of 43 COOH-terminal amino acids was engineered by cDNA-based deletion mutagenesis. This cDNA (HIR delta CT), as well as cDNA encoding the complete receptor (HIRc) was transfected into Rat 1 fibroblasts. Cells expressing 6.4 X 10(3) and 1.25 X 10(6) normal receptors and 2.5 X 10(5) HIR delta CT receptors, as well as control Rat 1 fibroblasts were selected for further analysis. All cell lines exhibited insulin binding of similar affinity. Partial tryptic digestion and immunoprecipitation by region-specific antibodies verified that the HIR delta CT receptors were truncated at the COOH terminus. Purified HIRc and HIR delta CT receptors underwent autophosphorylation with similar insulin and ATP sensitivity, although the HIR delta CT receptors were slightly more active in the absence of insulin. Transfected HIRc and HIR delta CT receptors undergo endocytosis in a normal fashion. Insulin internalization and degradation in both HIRc and HIR delta CT cells is increased in proportion to receptor number. Intracellular insulin processing, degradation, and release were qualitatively comparable among the transfected cell lines. Complete and truncated receptors internalize, recycle, and down-regulate normally. We conclude the following: 1) the COOH-terminal portion of the insulin receptor is not necessary for partial autophosphorylation or endocytosis; 2) following internalization the intracellular itinerary of the receptor and ligand appear normal with the truncated receptor; and 3) truncation of the COOH terminus does not impair recycling of the receptor or retroendocytosis of internalized ligand.     

Osteogenesis imperfecta: the audiological phenotype lacks correlation with the genotype

Background

Neurofibromatosis 1 (NF1), a common autosomal dominant disorder, was shown in one study to be associated with a 15-year decrease in life expectancy. However, data on mortality in NF1 are limited. Our aim was to evaluate mortality in a large retrospective cohort of NF1 patients seen in France between 1980 and 2006.

Methods

Consecutive NF1 patients referred to the National French Referral Center for Neurofibromatoses were included. The standardized mortality ratio (SMR) with its 95% confidence interval (CI) was calculated as the ratio of observed over expected numbers of deaths. We studied factors associated with death and causes of death.

Results

Between 1980 and 2006, 1895 NF1 patients were seen. Median follow-up was 6.8 years (range, 0.4-20.6). Vital status was available for 1226 (65%) patients, of whom 1159 (94.5%) survived and 67 (5.5%) died. Overall mortality was significantly increased in the NF1 cohort (SMR, 2.02; CI, 1.6-2.6; P < 10^-4). The excess mortality occurred among patients aged 10 to 20 years (SMR, 5.2; CI, 2.6-9.3; P < 10^-4) and 20 to 40 years (SMR, 4.1; 2.8-5.8; P < 10^-4). Significant excess mortality was found in both males and females. In the 10-20 year age group, females had a significant increase in mortality compared to males (SMR, 12.6; CI, 5.7-23.9; and SMR, 1.8; CI, 0.2-6.4; respectively). The cause of death was available for 58 (86.6%) patients; malignant nerve sheath tumor was the main cause of death (60%).

Conclusions

We found significantly increased SMRs indicating excess mortality in NF1 patients compared to the general population. The definitive diagnosis of NF1 in all patients is a strength of our study, and the high rate of death related to malignant transformation is consistent with previous work. The retrospective design and hospital-based recruitment are limitations of our study. Mortality was significantly increased in NF1 patients aged 10 to 40 years and tended to be higher in females than in males.     

Immune responses to stress after stress management training in patients with rheumatoid arthritis

Introduction

Psychological stress may alter immune function by activating physiological stress pathways. Building on our previous study, in which we report that stress management training led to an altered self-reported and cortisol response to psychological stress in patients with rheumatoid arthritis (RA), we explored the effects of this stress management intervention on the immune response to a psychological stress task in patients with RA.

Methods

In this study, 74 patients with RA, who were randomly assigned to either a control group or a group that received short stress management training, performed the Trier Social Stress Test (TSST) 1 week after the intervention and at a 9-week follow-up. Stress-induced changes in levels of key cytokines involved in stress and inflammatory processes (for example, interleukin (IL)-6 and IL-8) were assessed.

Results

Basal and stress-induced cytokine levels were not significantly different in patients in the intervention and control groups one week after treatment, but stress-induced IL-8 levels were lower in patients in the intervention group than in the control group at the follow-up assessment.

Conclusions

In line with our previous findings of lower stress-induced cortisol levels at the follow-up of stress management intervention, this is the first study to show that relatively short stress management training might also alter stress-induced IL-8 levels in patients with RA. These results might help to determine the role of immunological mediators in stress and disease.

Trial registration

The Netherlands National Trial Register (NTR1193)     

Quantitative Proteomic and Functional Analysis of Liver Mitochondria from High Fat Diet (HFD) Diabetic Mice

Insulin resistance plays a major role in the development of type 2 diabetes and obesity and affects a number of biological processes such as mitochondrial biogenesis. Though mitochondrial dysfunction has been linked to the development of insulin resistance and pathogenesis of type 2 diabetes, the precise mechanism linking the two is not well understood. We used high fat diet (HFD)-induced obesity dependent diabetes mouse models to gain insight into the potential pathways altered with metabolic disease, and carried out quantitative proteomic analysis of liver mitochondria. As previously reported, proteins involved in fatty acid oxidation, branched chain amino acid degradation, tricarboxylic acid cycle, and oxidative phosphorylation were uniformly up-regulated in the liver of HFD fed mice compared with that of normal diet. Further, our studies revealed that retinol metabolism is distinctly down-regulated and the mitochondrial structural proteins—components of mitochondrial inter-membrane space bridging (MIB) complex (Mitofilin, Sam50, and ChChd3), and Tim proteins—essential for protein import, are significantly up-regulated in HFD fed mice. Structural and functional studies on HFD and normal diet liver mitochondria revealed remodeling of HFD mitochondria to a more condensed form with increased respiratory capacity and higher ATP levels compared with normal diet mitochondria. Thus, it is likely that the structural remodeling is essential to accommodate the increased protein content in presence of HFD: the mechanism could be through the MIB complex promoting contact site and crista junction formation and in turn facilitating the lipid and protein uptake.Obesity has become a global epidemic and in the United States alone more than one third of adults (34%) are obese and over 11% of the population over the age of 20 are diabetic (1, 2). Even though the precise mechanisms causing obesity are still being determined, it is well established that obesity induces insulin resistance leading to the pathogenesis of type 2 diabetes (T2D)¹ (3, 4). Insulin resistance has been implicated in multiple organ damage such as liver, skeletal muscle, and adipose tissues (5). This is in view of the fact that cellular glucose homeostasis is tightly regulated by insulin secretion from the pancreatic β-cells and glucose uptake by muscle and output by liver. Thus, failure of insulin secretion by pancreatic β-cells to compensate for insulin resistance results in hyperglycemia (6, 7) and uncontrolled hyperglycemia has the potential to negatively impact a number of organ systems.Mitochondrial dysfunction has been thought to play a critical role in insulin resistance and T2D (8–11), and the role of mitochondria in insulin resistance is highly tissue specific. Despite this the mechanisms of action is controversial: in skeletal muscle, oxidative metabolism of lipids is reduced in T2D patients (11, 12). However, it has been reported that carnitine palmitoyl transferase (CPT) activity is decreased or long-chain acyl-CoA dehydrogenase (LACD) is deficient, and these lead to an accumulation of intracellular lipids and insulin resistance in insulin-targeting cells (13). These excess metabolites eventually cause the decrease in glucose transport and other events downstream of insulin receptor signaling (14). In adipose tissue, it has been shown that adiponectin expression and mitochondrial content in obese db/db mice were reduced (15). One of the favorable effects of adiponectin is the improvement of insulin action (16) and it has been reported that mitochondrial dysfunction in adipose tissue explains decreased adiponectin synthesis in obesity (15). In contrast to muscle, coordinated up-regulation of oxidative phosphorylation (OXPHOS) subunits in obese liver has been previously reported at mRNA (17) and protein (18) levels. Liver mitochondria also showed significant up-regulation of beta-oxidation, branched chain amino acid degradation, pyruvate metabolism, TCA cycle, and apoptotic pathways (18, 19).In the fasting state, blood glucose levels are maintained by liver regulated gluconeogenesis. T2D is characterized by defects in insulin''s ability to inhibit gluconeogenesis. Hepatic insulin resistance and impaired fatty acid oxidation have been the major contributors for the development of hepatic steatosis and the progression of nonalcoholic fatty liver disease (NAFLD) (20). Mitochondrial abnormalities have also been associated with NAFLD, and hence liver insulin sensitivity and the potential relationship with mitochondria appear to be crucial.In this study, we used a proteomic approach to investigate the biological pathways leading to insulin resistance in T2D in the liver and analyzed the protein expression profiles from mitochondria of mice fed on a high fat diet (HFD) for 30 weeks. Increased insulin resistance, elevated plasma insulin and impaired glucose tolerance were observed in mice after 16 weeks of HFD (21). Consistent with previous studies, liver mitochondria showed significant up-regulation of oxidation-phosphorylation (OXPHOS), beta-oxidation, branched chain amino acid degradation, pyruvate metabolism, TCA cycle, and apoptotic pathways (18, 19). Additionally, in our study we show that retinol metabolism was consistently down-regulated, and the proteins involved in the protein import into mitochondria, mitochondrial biogenesis and regulation of crista morphology are up-regulated in HFD versus normal diet (ND) liver mitochondria. We further examined the ultrastructure of the mitochondria from the liver and show that under HFD conditions the mitochondria are often condensed with a remodeling of crista and increased number of cristae. We suggest that the structural change in cristae is likely to be a necessary event to accommodate the increased OXPHOS gene expression and other proteins that are residents of the inner membrane. The remodeling of cristae is also accompanied by increased mitofilin expression and its interacting proteins, which include ChChd3 and OPA1, that are critical for cristae organization. Finally our oxygen consumption experiment showed increased activities of complex I and complex II in liver mitochondria of HFD versus ND mice and our ATP assay confirmed increased ATP production in HFD versus ND mice.