NMR Structural and Biophysical Analysis of the Disease-Linked Inner Mitochondrial Membrane Protein MPV17

MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.     

Cardiac myoglobin deficit has evolved repeatedly in teleost fishes

Myoglobin (Mb) is the classic vertebrate oxygen-binding protein present in aerobic striated muscles. It functions principally in oxygen delivery and provides muscle with its characteristic red colour. Members of the Antarctic icefish family (Channichthyidae) are widely thought to be extraordinary for lacking cardiac Mb expression, a fact that has been attributed to their low metabolic rate and unusual evolutionary history. Here, we report that cardiac Mb deficit, associated with pale heart colour, has evolved repeatedly during teleost evolution. This trait affects both gill- and air-breathing species from temperate to tropical habitats across a full range of salinities. Cardiac Mb deficit results from total pseudogenization in three-spined stickleback and is associated with a massive reduction in mRNA level in two species that evidently retain functional Mb. The results suggest that near or complete absence of Mb-assisted oxygen delivery to heart muscle is a common facet of teleost biodiversity, even affecting lineages with notable oxygen demands. We suggest that Mb deficit may affect how different teleost species deal with increased tissue oxygen demands arising under climate change.     

Free Radicals and Carcinogenesis

The role of free radicals and active states of oxygen in human cancer is as yet unresolved. Various lines of evidence provide strong but inferential evidence that free radical reactions can be of crucial importance in certain carcinogenic mechanisms. A central point in considering free radical reactions in carcinogenesis is that human cancer is really a group of highly diverse diseases for which the initial causation and the progression to clinical disease occur through a wide variety of mechanisms. Furthermore, for many human cancers it appears that there are alternate pathways capable of tumor initiation and tumor progression. While for certain of these pathways free radical reactions appear necessary, it is unlikely that there are human cancers for which free radicals, or any other mechanism, are sufficient for the entire processbeginning with the genetic alteration leading to a somatic mutation and eventually resulting in clinically overt disease. It is crucial that we view free radical reactions as aong a panoply of mechanisms leading to human cancer, and consider research about the role of free radicals in cancer as opportunities to prevent the initiation or progression of human cancer.     

The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M₄₁₂) and light-induced proton release under steady-state conditions in aqueous suspensions of ‘purple membrane’ derived from Halobacterium halobium. The decay of M₄₁₂ was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M₄₁₂ which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M₄₁₂ was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M₄₁₂ without affecting the level of M₄₁₂ which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca²⁺ and their antagonists La³⁺ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.     

Cyclo(His-Pro) up-regulates heme oxygenase 1 via activation of Nrf2-ARE signalling

Paraquat (1,1'-dimethyl-4,4'-bipyridinium), a widely used non-selective herbicide, is a redox cycling agent with adverse effects on dopamine systems. Epidemiological data have shown that exposure to paraquat is one of the several risk factors for Parkinson's disease. We have already shown that cyclo(His-Pro), an endogenous cyclic dipeptide produced by the cleavage of the thyrotropin releasing hormone, has a cytoprotective effect through a mechanism involving Nrf2 activation that decreases production of reactive oxygen species and increases glutathione synthesis. Using primary neuronal cultures and PC12 cells as targets of paraquat neurotoxicity, we addressed whether and how cyclo(His-Pro) causes cellular protective response against paraquat-mediated cell death. We found that cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion by up-regulating Nrf2 gene expression, triggering its nuclear accumulation and activating the expression of heme oxygenase1. These protective effects were abolished by RNA interference-mediated Nrf2 knock down whereas were unaffected by RNA interference-mediated Keap1 knock down. Inhibition of heme oxygenase activity decreased cyclo(His-Pro)-induced neuroprotection. These results suggest that cyclo(His-Pro), acting as a selective activator of the brain modulable Nrf2 pathway, may be a promising candidate as neuroprotective agent that act through induction of phase II genes.     

Oxygen: a master regulator of pancreatic development?

Beyond its role as an electron acceptor in aerobic respiration, oxygen is also a key effector of many developmental events. The oxygen‐sensing machinery and the very fabric of cell identity and function have been shown to be deeply intertwined. Here we take a first look at how oxygen might lie at the crossroads of at least two of the major molecular pathways that shape pancreatic development. Based on recent evidence and a thorough review of the literature, we present a theoretical model whereby evolving oxygen tensions might choreograph to a large extent the sequence of molecular events resulting in the development of the organ. In particular, we propose that lower oxygenation prior to the expansion of the vasculature may favour HIF (hypoxia inducible factor)‐mediated activation of Notch and repression of Wnt/β‐catenin signalling, limiting endocrine cell differentiation. With the development of vasculature and improved oxygen delivery to the developing organ, HIF‐mediated support for Notch signalling may decline while the β‐catenin‐directed Wnt signalling is favoured, which would support endocrine cell differentiation and perhaps exocrine cell proliferation/differentiation.     

Aging-related changes in the sensitivity of the system of respiratory control and the intensity of free-radical processes in humans

Results of a comparative study of the sensitivity of the system of respiratory control to increases in the CO₂ concentration and the intensity of free-radical processes in young and elderly subjects are described. It is shown that normal (natural) aging is accompanied by a decrease in the sensitivity of the respiratory system to hypercapnic stimulation and a parallel significant decrease in the activity of catalase in the blood of examined subjects. Mechanisms responsible for the modifications of the sensitivity of the system of respiratory control to hypercapnia are discussed; these shifts can be at least partly related to changes in the intensity of production of free radicals observed in elderly subjects. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 53–57, January–February, 2008.     

Pollen-based biome reconstruction for southern Europe and Africa 18,000 yr bp

Pollen data from 18,000 ¹⁴C yr bp were compiled in order to reconstruct biome distributions at the last glacial maximum in southern Europe and Africa. Biome reconstructions were made using the objective biomization method applied to pollen counts using a complete list of dryland taxa wherever possible. Consistent and major differences from present‐day biomes are shown. Forest and xerophytic woods/scrub were replaced by steppe, both in the Mediterranean region and in southern Africa, except in south‐western Cape Province where fynbos (xerophytic scrub) persisted. Sites in the tropical highlands, characterized today by evergreen forest, were dominated by steppe and/or xerophytic vegetation (cf. today’s Ericaceous belt and Afroalpine grassland) at the last glacial maximum. Available data from the tropical lowlands are sparse but suggest that the modern tropical rain forest was largely replaced by tropical seasonal forest while the modern seasonal or dry forests were encroached on by savanna or steppe. Montane forest elements descended to lower elevations than today.     

The Everyday Life Context of Increasing Energy Demands: Time Use Survey Data in a Decomposition Analysis

Industrial ecologists have modeled with precision the material foundations of industrial systems, but given less attention to the demand for products and the drivers of structural changes in these systems. This article suggests that time use data complement data on monetary expenditure and can be used to elucidate the everyday life context in which the changes in the economy take place. It builds upon the claim that goods are not direct sources of utility, but enter specific household activities as inputs. A second argument for the proposed approach is that it can be used to introduce and foster human agency in analyses of production systems. The article uses Finnish time use survey data, consumption expenditure data, and data on the sectoral energy intensities of financial output in the Finnish economy. First, a measure of the energy intensity of activities is derived by relating consumer time use and the required direct and indirect energy requirements. Second, the results include a decomposition of changes in the energy requirements of private consumption in Finland during the 1990s. It is shown that although the same activities on average require increasing energy inputs per unit of time, Finns have simultaneously changed the structure of their everyday life toward less energy-intensive activities.