Exploring the molecular basis of heme coordination in human neuroglobin

Neuroglobin (Ngb), a recently discovered ancient heme protein, presents the typical globin fold and is around 20% identical to myoglobin (Mb). In contrast with Mb, however, its heme is hexacoordinated (6c). It is expressed in the nervous system and has been the subject of numerous investigations in the last years, but its function is still unclear. The proposed roles include oxygen transport, reactive oxygen species (ROS) detoxification, hypoxia protection, and redox state sensing. All proposed functions require distal histidine dissociation from the heme to yield a reactive iron. With the aim of understanding the 6c to 5c transition, we have performed molecular dynamics simulations for ferrous Ngb in the 6c, 5c, and oxy states. We also computed free energy profiles associated with the transition employing an advanced sampling technique. Finally, we studied the effect of the redox state of CysCD7 and CysD5, which are known to form a disulfide bridge. Our results show that protein oxidation promotes a stabilization of the pentacoordinated species, thus favoring the protein to adopt the more reactive state and supporting the existence of a molecular mechanism whereby O2 would be released under hypoxic conditions, thereby suggesting an O(2) storage function for Ngb. Taken together, our results provide structural information not available experimentally which may shed light on the protein proposed functions, particularly as a redox sensor.     

Analysis of Site Frequency Spectra from Arabidopsis with Context-Dependent Corrections for Ancestral Misinference

Previous studies have shown that the pattern of single nucleotide polymorphism (SNP) in Arabidopsis (Arabidopsis thaliana) deviates from the distribution expected under a neutral model. Here, we test whether or not ancestral misinference could explain this deviation. We start by showing that there are significant and complex influences of context on mutation dynamics as inferred from SNP frequency, in Arabidopsis, and compare the results to observations about context dependency that have been made on a previous analysis of a maize (Zea mays) SNP dataset. The data concerning heterogeneity across sites are then used to make corrections for ancestral misinference in a context-dependent manner. Using Arabidopsis lyrata to infer the ancestral state for SNPs, we show that the resulting unfolded site frequency spectrum (SFS) in Arabidopsis is skewed toward sites with high frequency derived nucleotides. Sites are also partitioned into two general functional classes, second codon position and 4-fold degenerate sites. These two classes show different SFS; although both show an overrepresentation of high frequency derived sites, low frequency derived sites are vastly overrepresented at the second codon position, but significantly underrepresented at 4-fold degenerate sites. We find that these results are robust to corrections for ancestral misinference, even when context-dependent variation in mutation properties is taken into consideration. The data suggest that, in addition to purifying selection, complex demographic events and/or linked positive selection need to be invoked to explain the SFS, and they highlight the importance of sequence context in analyses of genome-wide variation.Analyses of site frequency spectra (SFS) from single nucleotide polymorphism (SNP) datasets provide a powerful method for making inferences about selection (Akashi, 1999; Bustamante et al., 2001; Hernandez et al., 2007). The allele frequency distribution expected under a neutral model (Tajima, 1989) can be applied to datasets for which an outgroup is available by unfolding the distribution using the assumption of parsimony. Deviation of this distribution from the neutral model provides insights about the role of selection or demographics; an overabundance of high frequency derived sites is frequently attributed to either recurrent positive selection (Bustamante et al., 2001; Caicedo et al., 2007), a population bottleneck (Caicedo et al., 2007), or hidden population substructure (Wakeley and Aliacar, 2001; Hernandez et al., 2007), whereas an excess of low frequency derived sites is commonly explained as a result of constraining selection or a recent population expansion (Slatkin and Hudson, 1991; Hernandez et al., 2007).Arabidopsis (Arabidopsis thaliana) represents one of the most intensively studied model organisms for molecular population genetics, and several genome-scale patterns of nucleotide variation have been generated (Nordborg et al., 2005; Schmid et al., 2005; Borevitz et al., 2007; Clark et al., 2007). These studies have shown evidence for genome-wide departures from a standard neutral population genetic model assuming constant population size. One recurring pattern is that minor allele frequencies tend to be skewed such that there is an excess of rare variants across the genome (Nordborg et al., 2005; Schmid et al., 2005). This pattern has typically been interpreted as evidence for population expansion, although other aspects of the genome-wide data, including a high variance in diversity across loci (Nordborg et al., 2005), appear inconsistent with a simple model of population growth. Furthermore, amino acid substitutions typically show a larger excess of rare variants (Foxe et al., 2008), suggestive of weak purifying selection across the genome.One limitation with these analyses is that outgroup data have rarely been available, restricting the ability to infer the derived frequency spectrum and thus distinguish new low frequency mutations from high frequency derived variants. Instead, these analyses implicitly rely on the theoretical prediction that the probability that an allele is ancestral is equal to its frequency (Watterson and Guess, 1977). In principle, the polarized frequency spectrum should provide considerably more information on the genome-wide patterns of variation and more power to infer the direction and strength of selection (Sawyer and Hartl, 1992; Akashi, 1999). However, a potential difficulty with the use of an outgroup to infer the ancestral and derived states at a given site is that the outgroup state is typically taken as ancestral under a parsimony assumption. This means that parallel changes could result in a misinference of the ancestral state, and this would generally lead to a skew toward sites with a high frequency of the derived state and, therefore, a potential for generating a spurious signature of positive selection or demographic effect (Baudry and Depaulis, 2003; Hernandez et al., 2007). Furthermore, given differences in effective mutation rates across different classes of sites, there may be biased rates of ancestral misinference, which can also lead to problems when inferring the strength of selection on different types of substitution. Given this potentially confounding effect of ancestral misinference, methods have been proposed to correct the SFS (e.g. Baudry and Depaulis, 2003; Hernandez et al., 2007).Any correction for ancestral misinference must be based on an adequate substitution model. In the case of plant genomes, including the maize (Zea mays) nuclear genome, it is well established that relative mutation rates vary significantly across sites as a function of context or the composition of surrounding nucleotides (Morton, 1995, 2003; Morton et al., 2006; Moore and Stevens, 2008) and similar context dependency has been observed in other genomes (Blake et al., 1992; Hess et al., 1994; Krawczak et al., 1998; Zhao and Boerwinkle, 2002). One prominent feature of context dependency is the CpG effect, or an increased rate of transitions at CG dinucleotides as a result of the relatively rapid deamination of methylated cytosines at many such sites (Bulmer, 1986; Zhao and Boerwinkle, 2002; Morton et al., 2006). More complex patterns of context dependency have also been observed in nuclear DNA of maize, where it has been shown that transition and transversion rates are significantly influenced by local and regional composition, but in different manners, and that the rate of mutation of GC and AT base pairs are affected differently by context (Morton et al., 2006).When complex context dependency exists, correcting for ancestral misinference would require that site context be taken into consideration (Hernandez et al., 2007). Therefore, we begin by analyzing heterogeneity across sites in Arabidopsis as a function of context. We find that mutation dynamics are influenced in a complex manner by both composition of flanking nucleotides and regional A+T content. These findings are compared to the context effects that have been observed in maize (Morton et al., 2006). We then analyze the unfolded SFS, with Arabidopsis lyrata as the outgroup, using the method of Baudry and Depaulis (2003) to account for ancestral misinference. To account for the influence of context on mutation dynamics, sites are partitioned by the number of flanking A/T base pairs because this was found to be a major contributing factor to context effects. Sites were also partitioned by codon position and degeneracy to account, approximately, for functional effects. An SFS was then generated for sites within each of the separate partitions and each spectrum was corrected using mutation parameters for that partition.We find that the excess of high frequency sites cannot be explained by ancestral misinference. In addition, second codon position sites show an excess of low frequency sites and 4-fold degenerate sites show a significant deficit of low frequency sites; both of these features remain after the correction. We suggest that complex demographic history and/or the action of positive selection have had a major effect on genome-wide patterns of variation, and we confirm the predominance of slightly deleterious amino acid polymorphisms in the Arabidopsis genome.     

The heme-binding protein HbpS regulates the activity of the <Emphasis Type="Italic">Streptomyces reticuli</Emphasis> iron-sensing histidine kinase SenS in a redox-dependent manner

The SenS/SenR system of Streptomyces reticuli regulates the expression of the redox regulator FurS, the catalase-peroxidase CpeB and the heme-binding protein HbpS. SenS/SenR is also proposed to participate in sensing redox changes, mediated by HbpS. Here, we show in vitro that heme-free HbpS represses the autokinase activity of SenS; whereas hemin-treated HbpS considerably enhances SenS autophosphorylation under redox conditions using either H₂O₂ or DTT. The presence of iron ions alone or in combination with H₂O₂ or DTT also leads to significantly increased phosphorylation levels of SenS. Further comparative physiological studies using the S. reticuli WT, a S. reticuli hbpS mutant and a S. reticuli senS-senR mutant corroborates the importance of HbpS and the SenS/SenR system for resistance against high concentrations of iron ions and hemin in vivo. Hence SenS/SenR and HbpS act in concert as a novel three-component system which detects redox stress, mediated by iron ions and heme.     

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples

Background  

Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C₄(RP)-LC protein separation) followed by C₁₈(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.     

Nuclear ataxia-telangiectasia mutated (ATM) mediates the cellular response to DNA double strand breaks in human neuron-like cells

The protein kinase ATM (ataxia-telangiectasia mutated) activates the cellular response to double strand breaks (DSBs), a highly cytotoxic DNA lesion. ATM is activated by DSBs and in turn phosphorylates key players in numerous damage response pathways. ATM is missing or inactivated in the autosomal recessive disorder ataxia-telangiectasia (A-T), which is characterized by neuronal degeneration, immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. The predominant symptom of A-T is a progressive loss of movement coordination due to ongoing degeneration of the cerebellar cortex and peripheral neuropathy. A major deficiency in understanding A-T is the lack of information on the role of ATM in neurons. It is unclear whether the ATM-mediated DSB response operates in these cells similarly to proliferating cells. Furthermore, ATM was reported to be cytoplasmic in neurons and suggested to function in these cells in capacities other than the DNA damage response. Recently we obtained genetic molecular evidence that the neuronal degeneration in A-T does result from defective DNA damage response. We therefore undertook to investigate this response in a model system of human neuron-like cells (NLCs) obtained by neuronal differentiation in culture. ATM was largely nuclear in NLCs, and their ATM-mediated responses to DSBs were similar to those of proliferating cells. Knocking down ATM did not interfere with neuronal differentiation but abolished ATM-mediated damage responses in NLCs. We concluded that nuclear ATM mediates the DSB response in NLCs similarly to in proliferating cells. Attempts to understand the neurodegeneration in A-T should be directed to investigating the DSB response in the nervous system.     

Tire-breeding mosquitoes of public health importance along an urbanisation gradient in Buenos Aires, Argentina

Used vehicle tires are a source of mosquito vectors and a means of their introduction and expansion. With the aim of assessing the effects of urbanisation on the main mosquito vectors in temperate Argentina, the infestation levels of Aedes aegypti (L.) and Culex pipiens L. were studied in used tires from highly urbanised cities to low-urbanised small towns in Buenos Aires. Immatures of both species accounted for 96% of the 9,722 individuals collected; the total individuals collected represented seven species. The percentage of water-filled tires containing mosquitoes [container index (CI)] was 33% and the percentage of infested sites [site index (SI)] was 65.2%. These indexes decreased significantly from low to high urbanisation levels for both mosquito species. The relative abundance (RA) of Ae. aegypti immatures was slightly higher toward large cities, but showed no difference for Cx. pipiens. The CI of shaded tires was significantly higher than the CI of exposed tires for both mosquito species. There was no difference in RA values between shaded and sunlit tires. The CI and the SI were highest during the summer across the urbanisation levels, except for Cx. pipiens, which continued to increase during the autumn in small towns. Results related to urbanisation gradient, sunlit exposure and seasonality are discussed.     

Protein and DNA destabilization by osmolytes: the other side of the coin

Osmolytes are naturally occurring small molecules accumulated intracellularly to protect organisms from various denaturing stresses. Similar to the two faces of a coin, several of these osmolytes are stabilizing and destabilizing proteins depending on the concentrations and/or solvent conditions. For example, the well known stabilizing osmolyte, trehalose destabilizes some proteins at high concentration and/or high pH. In spite of the fact that destabilizing aspects of osmolytes can modulate many cellular processes including regulation of protein homeostasis (proteostasis), protein-protein interaction, and protein-DNA interaction, researchers have mostly focused on the stabilizing aspects of osmolytes. Thus, it is important to look into both aspects of osmolytes to determine their precise role under physiological conditions. In this article, we have discussed both stabilizing and destabilizing/denaturant aspects of osmolytes to uncover both sides of the coin.     

Structural model for p75(NTR)-TrkA intracellular domain interaction: a combined FRET and bioinformatics study

Nerve growth factor (NGF) is a member of the neurotrophins, which are important regulators of embryonic development and adult function in the vertebrate nervous systems. The signaling elicited by NGF regulates diverse activities, including survival, axon growth, and synaptic plasticity. NGF action is mediated by engagement with two structurally unrelated transmembrane receptors, p75(NTR) and TrkA, which are co-expressed in a variety of cells. The functional interactions of these receptors have been widely demonstrated and include complex formation, convergence of signaling pathways, and indirect interaction through adaptor proteins. Each domain of the receptors was shown to be important for the formation of TrkA and p75(NTR) complexes, but only the intramembrane and transmembrane domains seemed to be crucial for the creation of high-affinity binding sites. However, whether these occur through a physical association of the receptors is unclear. In the present work, we demonstrate by F?rster resonance energy transfer that p75(NTR) and TrkA are physically associated through their intracellular (IC) domains and that this interaction occurs predominantly at the cell membrane and prior to NGF stimulation. Our data suggest that there is a pool of receptors dimerized before NGF stimulus, which could contribute to the high-affinity binding sites. We modeled the three-dimensional structure of the TrkA IC domain by homology modeling, and with this and the NMR-resolved structure of p75(NTR), we modeled the heterodimerization of TrkA and p75(NTR) by docking methods and molecular dynamics. These models, together with the results obtained by F?rster resonance energy transfer, provide structural insights into the receptors' physical association.