Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import

The Tim9–Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9–Tim10 hexameric assembly determined to 2.5 Å and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.     

Mouse Models of Polyglutamine Diseases: Review and Data Table. Part I

Polyglutamine (polyQ) disorders share many similarities, such as a common mutation type in unrelated human causative genes, neurological character, and certain aspects of pathogenesis, including morphological and physiological neuronal alterations. The similarities in pathogenesis have been confirmed by findings that some experimental in vivo therapy approaches are effective in multiple models of polyQ disorders. Additionally, mouse models of polyQ diseases are often highly similar between diseases with respect to behavior and the features of the disease. The common features shared by polyQ mouse models may facilitate the investigation of polyQ disorders and may help researchers explore the mechanisms of these diseases in a broader context. To provide this context and to promote the understanding of polyQ disorders, we have collected and analyzed research data about the characterization and treatment of mouse models of polyQ diseases and organized them into two complementary Excel data tables. The data table that is presented in this review (Part I) covers the behavioral, molecular, cellular, and anatomic characteristics of polyQ mice and contains the most current knowledge about polyQ mouse models. The structure of this data table is designed in such a way that it can be filtered to allow for the immediate retrieval of the data corresponding to a single mouse model or to compare the shared and unique aspects of many polyQ models. The second data table, which is presented in another publication (Part II), covers therapeutic research in mouse models by summarizing all of the therapeutic strategies employed in the treatment of polyQ disorders, phenotypes that are used to examine the effects of the therapy, and therapeutic outcomes.     

Cadmium Alters the Concentration of Fatty Acids in THP-1 Macrophages

Fatty acid composition of human immune cells influences their function. The aim of this study was to evaluate the effects of known toxicant and immunomodulator, cadmium, at low concentrations on levels of selected fatty acids (FAs) in THP-1 macrophages. The differentiation of THP-1 monocytes into macrophages was achieved by administration of phorbol myristate acetate. Macrophages were incubated with various cadmium chloride (CdCl₂) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM, and 2 μM CdCl₂. Fatty acids were extracted from samples according to the Folch method. The fatty acid levels were determined using gas chromatography. The following fatty acids were analyzed: long-chain saturated fatty acids (SFAs) palmitic acid and stearic acid, very long-chain saturated fatty acid (VLSFA) arachidic acid, monounsaturated fatty acids (MUFAs) palmitoleic acid, oleic acid and vaccenic acid, and n-6 polyunsaturated fatty acids (PUFAs) linoleic acid and arachidonic acid. Treatment of macrophages with very low concentrations of cadmium (5–200 nM) resulted in significant reduction in the levels of arachidic, palmitoleic, oleic, vaccenic, and linoleic acids and significant increase in arachidonic acid levels (following exposure to 5 nM Cd), without significant reduction of palmitic and stearic acid levels. Treatment of macrophages with the highest tested cadmium concentration (2 μM) produced significant reduction in the levels of all examined FAs: SFAs, VLSFA, MUFAs, and PUFAs. In conclusion, cadmium at tested concentrations caused significant alterations in THP-1 macrophage fatty acid levels, disrupting their composition, which might dysregulate fatty acid/lipid metabolism thus affecting macrophage behavior and inflammatory state.     

The new insight into extracellular NAD+ degradation-the contribution of CD38 and CD73 in calcific aortic valve disease

Nicotinamide adenine dinucleotide (NAD⁺) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto-enzymes in controlling adenine nucleotide–dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD⁺ and mononucleotide nicotinamide (NMN) in aortic valves and aorta fragments of CAVD patients and on the inner aortic surface of ecto-5′-nucleotidase knockout mice (CD73−/−). Human non-stenotic valves (n = 10) actively converted NAD⁺ and NMN via both CD73 and NAD⁺-glycohydrolase (CD38) according to our analysis with RP-HPLC and immunofluorescence. In stenotic valves (n = 50), due to reduced CD73 activity, NAD⁺ was degraded predominantly by CD38 and additionally by ALP and eNPP1. CAVD patients had significantly higher hydrolytic rates of NAD⁺ (0.81 ± 0.07 vs 0.56 ± 0.10) and NMN (1.12 ± 0.10 vs 0.71 ± 0.08 nmol/min/cm²) compared with controls. CD38 was also primarily engaged in human vascular NAD⁺ metabolism. Studies using specific ecto-enzyme inhibitors and CD73−/− mice confirmed that CD73 is not the only enzyme involved in NAD⁺ and NMN hydrolysis and that CD38 had a significant contribution to these pathways. Modifications of extracellular NAD⁺ and NMN metabolism in aortic valve cells may be particularly important in valve pathology and could be a potential therapeutic target.     

Purinergic modulation of glucose uptake into cultured rat podocytes: effect of diabetic milieu

Extracellular purines act via P1 and P2 receptors on podocytes and may influence on their function. This action may be modified under various (patho)physiological conditions leading to development of podocytopathy. Aim of study was to investigate effects of diabetic milieu, represented by high glucose concentration (HG, 30 mM glucose) on purinergic-induced changes of 2-deoxy-d-glucose (2-DG) uptake and on extracellular purines metabolism in cultured rat podocytes. Basal 2-DG uptake was 2.7-fold enhanced in HG compared to normal glucose concentration, NG (1271 ± 86 vs. 477 ± 37 nmol/h/mg protein, P < 0.001). ATP stimulated 2-DG uptake by 44 ± 4% and 29 ± 5% in NG and HG, respectively. ATP analogues, β, γ-methylene ATP and 2-methylthio ATP stimulated 2-DG uptake in range of 18–34% in NG and 16–17% in HG. Benzoylbenzoyl ATP increased 2-DG uptake about 24 ± 2% in NG however, its effect in HG reached 50 ± 1%. The antagonists of P2 receptors (suramin, reactive blue 2, PPADS) decreased basal 2-DG uptake in NG and HG; suramin and reactive blue 2 at average of 15 ± 4% in NG but in HG the effect was in following order: suramin 28 ± 3%; PPADS 20 ± 3% and RB-2 9 ± 0.9%. Extracellular adenosine concentration was higher in HG than in NG (0.48 ± 0.01 vs. 5.05 ± 0.39 μM, P < 0.05), however intracellular ATP content and extracellular ATP concentration were not affected. Neither ecto-ATPase nor ecto-5′-nucleotidase activities were affected in HG. In conclusion, diabetic milieu affects purinergic modulation of glucose transport into podocytes which may play a role in development of diabetic podocytopathy.     

Extracellular ATP through P2 receptors activates AMP-activated protein kinase and suppresses superoxide generation in cultured mouse podocytes

Podocytes are an important constituent of the glomerular filtration barrier. The function of these glomerular cells is affected by extracellular nucleotides through P2 receptors. The activation of P2 receptors may lead to the activation of NAD(P)H oxidase, the key enzyme in oxidative stress, with the intracellular pathways leading to intracellular ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This deregulation of the energy balance activates AMP-activated protein kinase (AMPK) to restore energy homeostasis. We investigated whether P2 receptor activation influences NAD(P)H oxidase-dependent rate of superoxide anion (O₂^•−) generation and AMPK activity in cultured mouse podocytes. The rate of O₂^•− generation was measured by chemiluminescence and changes in AMPK activity were determined by immunoblotting against AMPKα-Thr¹⁷²-P. The addition of 100 μM ATP induced a rapid and transient decrease in rate of O₂^•− generation and increased AMPK phosphorylation with maximal effects in the first minute (2.44 ± 0.09 versus 1.62 ± 0.06 nmol/mg protein/min, P < 0.05 and 0.64 ± 0.04 versus 0.97 ± 0.07, P < 0.05, respectively). Both parameters returned to control levels at 10 min. Suramin (300 μM, P2 receptor antagonist) and compound C (100 μM, AMPK inhibitor) completely, and STO-609 (25 μM, CaMKK-β inhibitor) partially, prevented ATP action in rate of O₂^•− generation and AMPK phosphorylation. Various ATP analogues (10 μM) mimicked the effects of ATP on rate of O₂^•− generation and AMPK phosphorylation. The data indicate that extracellular ATP, acting through P2 receptors upstream of CaMKK-β, modulates podocyte function through simultaneous effects on AMPK and NAD(P)H oxidase activities. This mechanism may play a role in restoring energy homeostasis after oxidative stress.     

Increased Lipid Peroxidation and Ascorbic Acid Utilization in Testis and Epididymis of Rats Chronically Exposed to Lead

The hypothesis has been recently presented that lead may exert its negative effect at least partially through the increase of reactive oxygen species (ROS) level in tissues. However, little is known about the influence of lead intoxication on equilibrium between generation and elimination of ROS in the male reproductive system. Sexually mature male Wistar rats were given ad libitum 1% of aqueous solution of lead acetate (PbAc) for 9 months. Significantly higher lead concentrations were found in blood [median 7.03 (Q25–Q75: 2.99–7.65) versus 0.18 (0.12–0.99) μg dl⁻¹, P < 0.01], caput epididymis [median 5.51 (Q25–Q75: 4.31–7.83) versus 0.51 (0.11–0.80) μg g⁻¹ d.m., P < 0.001], cauda epididymis [median 5.88 (Q25–Q75: 4.06–8.37) versus 0.61 (0.2 – 1.08) μg g⁻¹ d.m., P < 0.001] and testis [median 1.81 (Q25–Q75: 0.94–2.31) versus 0.17 (0.03–0.3) μg g⁻¹ d.m., P < 0.01] of lead-intoxicated rats when compared to the control. The concentration of ascorbyl radical, generated in vitro from l-ascorbic acid (present in tissues in vivo) was measured by means of Electron Paramagnetic Resonance (EPR) spectroscopy. The EPR signal of ascorbyl radical in caput epididymis, cauda epididymis, testis and liver of lead acetate-treated animals revealed a significant decrease by 53%, 45%, 40% and 69% versus control tissues, respectively. Plasma l-ascorbic acid content measured by high performance liquid chromatography (HPLC) method and total antioxidant status (TAS) measured by means of spectrophotometry were also significantly lower in the intoxicated versus control animals (28% and 21%, respectively). In the group exposed to lead the concentration of lipid peroxide in homogenates of the reproductive system organs was significantly elevated versus control group. It can be assumed that the lower EPR signal was caused by decreased tissue concentrations of l-ascorbic acid. The latter may have resulted from consumption of ascorbic acid for scavenging of ROS excess in tissues of animals chronically exposed to lead.