Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein.

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling ßD from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP.

Reverse and Forward Electron Flow-Induced H2O2 Formation Is Decreased in α-Ketoglutarate Dehydrogenase (α-KGDH) Subunit (E2 or E3) Heterozygote Knock Out Animals.

α-ketoglutarate dehydrogenase complex (KGDHc), or 2-oxoglutarate dehydrogenase complex (OGDHc) is a rate-limiting enzyme in the tricarboxylic acid cycle, that has been identified in neurodegenerative diseases such as in Alzheimer's disease. The aim of the present study was to establish the role of the KGDHc and its subunits in the bioenergetics and reactive oxygen species (ROS) homeostasis of brain mitochondria. To study the bioenergetic profile of KGDHc, genetically modified mouse strains were used having a heterozygous knock out (KO) either in the dihydrolipoyl succinyltransferase (DLST+/-) or in the dihydrolipoyl dehydrogenase (DLD+/-) subunit. Mitochondrial oxygen consumption, hydrogen peroxide (H2O2) production, and expression of antioxidant enzymes were measured in isolated mouse brain mitochondria. Here, we demonstrate that the ADP-stimulated respiration of mitochondria was partially arrested in the transgenic animals when utilizing α-ketoglutarate (α-KG or 2-OG) as a fuel substrate. Succinate and α-glycerophosphate (α-GP), however, did not show this effect. The H2O2 production in mitochondria energized with α-KG was decreased after inhibiting the adenine nucleotide translocase and Complex I (CI) in the transgenic strains compared to the controls. Similarly, the reverse electron transfer (RET)-evoked H2O2 formation supported by succinate or α-GP were inhibited in mitochondria isolated from the transgenic animals. The decrease of RET-evoked ROS production by DLST+/- or DLD+/- KO-s puts the emphasis of the KGDHc in the pathomechanism of ischemia-reperfusion evoked oxidative stress. Supporting this notion, expression of the antioxidant enzyme glutathione peroxidase was also decreased in the KGDHc transgenic animals suggesting the attenuation of ROS-producing characteristics of KGDHc. These findings confirm the contribution of the KGDHc to the mitochondrial ROS production and in the pathomechanism of ischemia-reperfusion injury.

The effect of Cyclophilin D depletion on liver regeneration following associating liver partition and portal vein ligation for staged hepatectomy.

AIM: Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) is a modification of two-stage hepatectomy profitable for patients with inoperable hepatic tumors by standard techniques. Unfortunately, initially poor postoperative outcome was associated with ALPPS, in which mitochondrial dysfunction played an essential role. Inhibition of cyclophilins has been already proposed to be efficient as a mitochondrial therapy in liver diseases. To investigate the effect of Cyclophilin D (CypD) depletion on mitochondrial function, biogenesis and liver regeneration following ALPPS a CypD knockout (KO) mice model was created. METHODS: Male wild type (WT) (n = 30) and CypD KO (n = 30) mice underwent ALPPS procedure. Animals were terminated pre-operatively and 24, 48, 72 or 168 h after the operation. Mitochondrial functional studies and proteomic analysis were performed. Regeneration rate and mitotic activity were assessed. RESULTS: The CypD KO group displayed improved mitochondrial function, as both ATP production (P < 0.001) and oxygen consumption (P < 0.05) were increased compared to the WT group. The level of mitochondrial biogenesis coordinator peroxisome proliferator-activated receptor γ co-activator 1-α (PGC1-α) was also elevated in the CypD KO group (P < 0.001), which resulted in the induction of the mitochondrial oxidative phosphorylation system. Liver growth increased in the CypD KO group compared to the WT group (P < 0.001). CONCLUSIONS: Our study demonstrates the beneficial effect of CypD depletion on the mitochondrial vulnerability following ALPPS. Based on our results we propose that CypD inhibition should be further investigated as a possible mitochondrial therapy following ALPPS.

The effects of physical prehabilitation: Improved liver regeneration and mitochondrial function after ALPPS operation in a rodent model.

BACKGROUND: To identify the role of physical prehabilitation (PP) in liver regeneration, mitochondrial function, biogenesis, and inflammatory response was investigated after associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) in a rodent model. METHODS: Male Wistar rats (n = 60) underwent ALPPS. Animals were divided (n = 30) to the physical prehabilitation group (PP) and sedentary group (S). The animals were exsanguinated before (0 hour) and 24, 48, 72, or 168 hours after the operation. Regeneration rate and proliferation index were assessed. Mitochondrial function, biogenesis, and inflammatory response were evaluated. RESULTS: Regeneration rate and Ki67 index were significantly increased in the PP group compared to the S group (P < .001). Due to the changes in oxidative capacity and ATP production rate, the P/O ratio of PP group compared to the S group was significantly increased (P < .05). PP group was characterized by accelerated mitochondrial biogenesis and less intense inflammatory response compared to the S group. CONCLUSIONS: To our knowledge, this is the first demonstration of the beneficial effects of PP on liver regeneration, mitochondrial function, biogenesis, and the inflammatory response after ALPPS.

Bioenergetic Impairment of Triethylene Glycol Dimethacrylate- (TEGDMA-) Treated Dental Pulp Stem Cells (DPSCs) and Isolated Brain Mitochondria are Amended by Redox Compound Methylene Blue †.

BACKGROUND: Triethylene glycol dimethacrylate (TEGDMA) monomers released from resin matrix are toxic to dental pulp cells, induce apoptosis, oxidative stress and decrease viability. Recently, mitochondrial complex I (CI) was identified as a potential target of TEGDMA. In isolated mitochondria supported by CI, substrates oxidation and ATP synthesis were inhibited, reactive oxygen species production was stimulated. Contrary to that, respiratory Complex II was not impaired by TEGDMA. The beneficial effects of electron carrier compound methylene blue (MB) are proven in many disease models where mitochondrial involvement has been detected. In the present study, the bioenergetic effects of MB on TEGDMA-treated isolated mitochondria and on human dental pulp stem cells (DPSC) were analyzed. METHODS: Isolated mitochondria and DPSC were acutely exposed to low millimolar concentrations of TEGDMA and 2 µM concentration of MB. Mitochondrial and cellular respiration and glycolytic flux were measured by high resolution respirometry and by Seahorse XF extracellular analyzer. Mitochondrial membrane potential was measured fluorimetrically. RESULTS: MB partially restored the mitochondrial oxidation, rescued membrane potential in isolated mitochondria and significantly increased the impaired cellular O2 consumption in the presence of TEGDMA. CONCLUSION: MB is able to protect against TEGDMA-induced CI damage, and might provide protective effects in resin monomer exposed cells.

Novel mitochondrial transition pore inhibitor N-methyl-4-isoleucine cyclosporin is a new therapeutic option in acute pancreatitis.

KEY POINTS: •Bile acids, ethanol and fatty acids affect pancreatic ductal fluid and bicarbonate secretion via mitochondrial damage, ATP depletion and calcium overload. •Pancreatitis-inducing factors open the membrane transition pore (mPTP) channel via cyclophilin D activation in acinar cells, causing calcium overload and cell death; genetic or pharmacological inhibition of mPTP improves the outcome of acute pancreatitis in animal models. •Here we show that genetic and pharmacological inhibition of mPTP protects mitochondrial homeostasis and cell function evoked by pancreatitis-inducing factors in pancreatic ductal cells. •The results also show that the novel cyclosporin A derivative NIM811 protects mitochondrial function in acinar and ductal cells, and it preserves bicarbonate transport mechanisms in pancreatic ductal cells. •We found that NIM811 is highly effective in different experimental pancreatitis models and has no side-effects. NIM811 is a highly suitable compound to be tested in clinical trials. ABSTRACT: Mitochondrial dysfunction plays a crucial role in the development of acute pancreatitis (AP); however, no compound is currently available with clinically acceptable effectiveness and safety. In this study, we investigated the effects of a novel mitochondrial transition pore inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), in AP. Pancreatic ductal and acinar cells were isolated by enzymatic digestion from Bl/6 mice. In vitro measurements were performed by confocal microscopy and microfluorometry. Preventative effects of pharmacological [cylosporin A (2 µm), NIM811 (2 µm)] or genetic (Ppif-/- /Cyp D KO) inhibition of the mitochondrial transition pore (mPTP) during the administration of either bile acids (BA) or ethanol + fatty acids (EtOH+FA) were examined. Toxicity of mPTP inhibition was investigated by detecting apoptosis and necrosis. In vivo effects of the most promising compound, NIM811 (5 or 10 mg kg-1 per os), were checked in three different AP models induced by either caerulein (10 × 50 µg kg-1 ), EtOH+FA (1.75 g kg-1 ethanol and 750 mg kg-1 palmitic acid) or 4% taurocholic acid (2 ml kg-1 ). Both genetic and pharmacological inhibition of Cyp D significantly prevented the toxic effects of BA and EtOH+FA by restoring mitochondrial membrane potential (Δψ) and preventing the loss of mitochondrial mass. In vivo experiments revealed that per os administration of NIM811 has a protective effect in AP by reducing oedema, necrosis, leukocyte infiltration and serum amylase level in AP models. Administration of NIM811 had no toxic effects. The novel mitochondrial transition pore inhibitor NIM811 thus seems to be an exceptionally good candidate compound for clinical trials in AP.

Ligand entry in human ileal bile acid-binding protein is mediated by histidine protonation.

Human ileal bile acid-binding protein (hI-BABP) has a key role in the intracellular transport of bile salts. To explore the role of histidine protonation in the binding process, the pH-dependence of bile salt binding and internal dynamics in hI-BABP was investigated using NMR spectroscopy and biophysical tools. Thermodynamic and kinetic measurements show an increase in the overall binding affinity and the association rate constant of the first binding step below the pKa of the histidines, suggesting that ligand binding is favoured by the protonated state. The overlap between residues exhibiting a high sensitivity to pH in their backbone amide chemical shifts and protein regions undergoing a global ms conformational exchange indicate a connection between the two processes. According to 15N NMR relaxation dispersion analysis, the slow motion is most pronounced at and above the pKa of the histidines. In agreement with the NMR measurements, MD simulations show a stabilization of the protein by histidine protonation. Hydrogen-bonding and van der Waals interactions mediating the flow of information between the C/D- and G/H-turn regions hosting the three histidines, suggest a complex way of pH-governed allosteric regulation of ligand entry involving a transition between a closed and a more open protein state.

Mutated SUCLG1 causes mislocalization of SUCLG2 protein, morphological alterations of mitochondria and an early-onset severe neurometabolic disorder.

Succinate-CoA ligase (SUCL) is a heterodimer consisting of an alpha subunit encoded by SUCLG1, and a beta subunit encoded by either SUCLA2 or SUCLG2 catalyzing an ATP- or GTP-forming reaction, respectively, in the mitochondrial matrix. The deficiency of this enzyme represents an encephalomyopathic form of mtDNA depletion syndromes. We describe the fatal clinical course of a female patient with a pathogenic mutation in SUCLG1 (c.626C > A, p.Ala209Glu) heterozygous at the genomic DNA level, but homozygous at the transcriptional level. The patient exhibited early-onset neurometabolic abnormality culminating in severe brain atrophy and dystonia leading to death by the age of 3.5 years. Urine and plasma metabolite profiling was consistent with SUCL deficiency which was confirmed by enzyme analysis and lack of mitochondrial substrate-level phosphorylation (mSLP) in skin fibroblasts. Oxygen consumption- but not extracellular acidification rates were altered only when using glutamine as a substrate, and this was associated with mild mtDNA depletion and no changes in ETC activities. Immunoblot analysis revealed no detectable levels of SUCLG1, while SUCLA2 and SUCLG2 protein expressions were largely reduced. Confocal imaging of triple immunocytochemistry of skin fibroblasts showed that SUCLG2 co-localized only partially with the mitochondrial network which otherwise exhibited an increase in fragmentation compared to control cells. Our results outline the catastrophic consequences of the mutated SUCLG1 leading to strongly reduced SUCL activity, mSLP impairment, mislocalization of SUCLG2, morphological alterations in mitochondria and clinically to a severe neurometabolic disease, but in the absence of changes in mtDNA levels or respiratory complex activities.

GABA, glutamine, glutamate oxidation and succinic semialdehyde dehydrogenase expression in human gliomas.

BACKGROUND: Bioenergetic characterisation of malignant tissues revealed that different tumour cells can catabolise multiple substrates as salvage pathways, in response to metabolic stress. Altered metabolism in gliomas has received a lot of attention, especially in relation to IDH mutations, and the associated oncometabolite D-2-hydroxyglutarate (2-HG) that impact on metabolism, epigenetics and redox status. Astrocytomas and oligodendrogliomas, collectively called diffuse gliomas, are derived from astrocytes and oligodendrocytes that are in metabolic symbiosis with neurons; astrocytes can catabolise neuron-derived glutamate and gamma-aminobutyric acid (GABA) for supporting and regulating neuronal functions. METHODS: Metabolic characteristics of human glioma cell models - including mitochondrial function, glycolytic pathway and energy substrate oxidation - in relation to IDH mutation status and after 2-HG incubation were studied to understand the Janus-faced role of IDH1 mutations in the progression of gliomas/astrocytomas. The metabolic and bioenergetic features were identified in glioma cells using wild-type and genetically engineered IDH1-mutant glioblastoma cell lines by metabolic analyses with Seahorse, protein expression studies and liquid chromatography-mass spectrometry. RESULTS: U251 glioma cells were characterised by high levels of glutamine, glutamate and GABA oxidation. Succinic semialdehyde dehydrogenase (SSADH) expression was correlated to GABA oxidation. GABA addition to glioma cells increased proliferation rates. Expression of mutated IDH1 and treatment with 2-HG reduced glutamine and GABA oxidation, diminished the pro-proliferative effect of GABA in SSADH expressing cells. SSADH protein overexpression was found in almost all studied human cases with no significant association between SSADH expression and clinicopathological parameters (e.g. IDH mutation). CONCLUSIONS: Our findings demonstrate that SSADH expression may participate in the oxidation and/or consumption of GABA in gliomas, furthermore, GABA oxidation capacity may contribute to proliferation and worse prognosis of gliomas. Moreover, IDH mutation and 2-HG production inhibit GABA oxidation in glioma cells. Based on these data, GABA oxidation and SSADH activity could be additional therapeutic targets in gliomas/glioblastomas.

Evaluation of mitochondrial function in chronic myofascial trigger points - a prospective cohort pilot study using high-resolution respirometry.

BACKGROUND: Myofascial trigger points (MTrPs) are hyperirritable areas in the fascia of the affected muscle, possibly related to mitochondrial impairment. They can result in pain and hypoxic areas within the muscle. This pilot study established a minimally invasive biopsy technique to obtain high-quality MTrP tissue samples to evaluate mitochondrial function via high-resolution respirometry. Secondary objectives included the feasibility and safety of the biopsy procedure. METHODS: Twenty healthy males participated in this study, 10 with a diagnosis of myofascial pain in the musculus (m.) trapezius MTrP (TTP group) and 10 with a diagnosis of myofascial pain in the m. gluteus medius (GTP group). Each participant had 2 muscle biopsies taken in one session. The affected muscle was biopsied followed by a biopsy from the m. vastus lateralis to be used as a control. Measurements of oxygen consumption were carried out using high-resolution respirometry. RESULTS: Mitochondrial respiration was highest in the GTP group compared to the TTP group and the control muscle whereas no differences were observed between the GTP and the control muscle. When normalizing respiration to an internal reference state, there were no differences between muscle groups. None of the participants had hematomas or reported surgical complications. Patient-reported pain was minimal for all 3 groups. All participants reported a low procedural burden. CONCLUSIONS: This pilot study used a safe and minimally invasive technique for obtaining biopsies from MTrPs suitable for high-resolution respirometry analysis of mitochondrial function. The results suggest that there are no qualitative differences in mitochondrial function of MTrPs of the trapezius and gluteus medius muscles compared to the vastus lateralis control muscle, implying that alterations of mitochondrial function do not appear to have a role in the development of MTrPs. TRIAL REGISTRATION: Registered as No. 20131128-850 at the Coordinating Center for Clinical Studies of the Medical University of Innsbruck, trial registration date: 28th November 2013 and retrospectively registered on 11th of October 2018 at ClinicalTrials.gov with the ID NCT03704311 .

Triethylene glycol dimethacrylate impairs bioenergetic functions and induces oxidative stress in mitochondria via inhibiting respiratory Complex I.

OBJECTIVES: Earlier studies demonstrated that dental resin monomers lower cellular viability and provoke oxidative stress. Reactive oxygen species (ROS) formation has a key role in triethylene glycol dimethacrylate (TEGDMA) induced adverse reactions. In the present study the effects of TEGDMA on mitochondrial functions were investigated to identify a direct molecular target for cytotoxicity. METHODS: Mitochondria were isolated from guinea pig brain. The most important bioenergetic parameters, oxygen consumption, membrane potential (ΔΨm), and ATP production were assessed. Mitochondrial H2O2 production and elimination and the NAD(P)H level reported on redox balance. RESULTS: Mitochondria were supported with respiratory substrates to be oxidized by either Complex I (CI) or Complex II (CII). ΔΨm was depolarized, respiration and ATP production was greatly diminished when applying CI substrates in the presence of TEGDMA. The same parameters remained essentially unaffected when CII substrate plus TEGDMA were applied. H2O2 production by mitochondria was significantly stimulated by TEGDMA in the presence of CI substrates. In the presence of TEGDMA mitochondrial elimination of exogenous H2O2 was impaired. When CII substrate supported the mitochondria in the absence of ADP the H2O2 generation was decreased. NADH autofluorescence results also demonstrated the inhibitory effect of TEGDMA on CI activity. SIGNIFICANCE: TEGDMA inhibits CI in the respiratory chain, which explains effects induced by TEGDMA on redox homeostasis, apoptotic and necrotic cell deaths described in previous studies. Identification of the molecular target of TEGDMA may influence the development of relevant biomaterials and may induce new therapeutic strategies to control the adverse effects of resin monomers.

Versatility of microglial bioenergetic machinery under starving conditions.

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.

Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid-binding protein.

Human ileal bile acid-binding protein (I-BABP) has a key role in the intracellular transport and metabolic targeting of bile salts. Similar to other members of the family of intracellular lipid-binding proteins (iLBPs), disorder-order transitions and local unfolding processes are thought to mediate ligand entry and release in human I-BABP. To gain insight into the stability of various protein regions, the temperature response of human I-BABP was investigated using NMR, CD and fluorescence spectroscopy, as well as molecular dynamics (MD) simulations. A joint analysis of NMR thermal melting and relaxation dispersion data indicates a complex pattern of internal dynamics with a dominating single barrier and a likely presence of rapidly exchanging conformational substates on both sides of the barrier. Moreover, our residue-specific analysis uncovers a partially unfolded U* state in which part of the helical region with three proximate ß-strands contains a substantial amount of residual structure, whereas several segments of the C-terminal half exhibit a high susceptibility to temperature elevation. Cluster analysis of atomic temperature responses indicates a thermodynamic coupling between distant protein sites including the bottom of the ß-barrel, the E-F region and part of the helical cap. MD simulations up to 1 µs show correlated motions in the same protein regions and together with the NMR data suggest a role for the highly dynamic D-E turn and E-F region in the initiation of unfolding. The response of human I-BABP to temperature elevation is discussed in the context of the folding/unfolding behaviour of different members of the iLBP family.

TP53 mutation hits energy metabolism and increases glycolysis in breast cancer.

Promising new hallmarks of cancer is alteration of energy metabolism that involves molecular mechanisms shifting cancer cells to aerobe glycolysis. Our goal was to evaluate the correlation between mutation in the commonly mutated tumor suppressor gene TP53 and metabolism. We established a database comprising mutation and RNA-seq expression data of the TCGA repository and performed receiver operating characteristics (ROC) analysis to compare expression of each gene between TP53 mutated and wild type samples. All together 762 breast cancer samples were evaluated of which 215 had TP53 mutation. Top up-regulated metabolic genes include glycolytic enzymes (e.g. HK3, GPI, GAPDH, PGK1, ENO1), glycolysis regulator (PDK1) and pentose phosphate pathway enzymes (PGD, TKT, RPIA). Gluconeogenesis enzymes (G6PC3, FBP1) were down-regulated. Oxygen consumption and extracellular acidification rates were measured in TP53 wild type and mutant breast cell lines with a microfluorimetric analyzer. Applying metabolic inhibitors in the presence and absence of D-glucose and L-glutamine in cell culture experiments resulted in higher glycolytic and mitochondrial activity in TP53 mutant breast cancer cell lines. In summary, TP53 mutation influences energy metabolism at multiple levels. Our results provide evidence for the synergistic activation of multiple hallmarks linking to these the mutation status of a key driver gene.

Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR.

Besides aiding digestion, bile salts are important signal molecules exhibiting a regulatory role in metabolic processes. Human ileal bile acid binding protein (I-BABP) is an intracellular carrier of bile salts in the epithelial cells of the distal small intestine and has a key role in the enterohepatic circulation of bile salts. Positive binding cooperativity combined with site selectivity of glycocholate and glycochenodeoxycholate, the two most abundant bile salts in the human body, make human I-BABP a unique member of the family of intracellular lipid binding proteins. Solution NMR structure of the ternary complex of human I-BABP with glycocholate and glycochenodeoxycholate reveals an extensive network of hydrogen bonds and hydrophobic interactions stabilizing the bound bile salts. Conformational changes accompanying bile salt binding affects four major regions in the protein including the C/D, E/F and G/H loops as well as the helical segment. Most of these protein regions coincide with a previously described network of millisecond time scale fluctuations in the apo protein, a motion absent in the bound state. Comparison of the heterotypic doubly ligated complex with the unligated form provides further evidence of a conformation selection mechanism of ligand entry. Structural and dynamic aspects of human I-BABP-bile salt interaction are discussed and compared with characteristics of ligand binding in other members of the intracellular lipid binding protein family. PROTEIN DATA BANK ACCESSION NUMBERS: The coordinates of the 10 lowest energy structures of the human I-BABP : GCDA : GCA complex as well as the distance restraints used to calculate the final ensemble have been deposited in the Brookhaven Protein Data Bank with accession number 2MM3.

Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage.

Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.

Temperature dependence of backbone dynamics in human ileal bile acid-binding protein: implications for the mechanism of ligand binding.

Human ileal bile acid-binding protein (I-BABP), a member of the family of intracellular lipid binding proteins plays a key role in the cellular trafficking and metabolic regulation of bile salts. The protein has two internal and, according to a recent study, an additional superficial binding site and binds di- and trihydroxy bile salts with positive cooperativity and a high degree of site-selectivity. Previously, in the apo form, we have identified an extensive network of conformational fluctuations on the millisecond time scale, which cease upon ligation. Additionally, ligand binding at room temperature was found to be accompanied by a slight rigidification of picosecond-nanosecond (ps-ns) backbone flexibility. In the current study, temperature-dependent (15)N NMR spin relaxation measurements were used to gain more insight into the role of dynamics in human I-BABP-bile salt recognition. According to our analysis, residues sensing a conformational exchange in the apo state can be grouped into two clusters with slightly different exchange rates. The entropy-enthalpy compensation observed for both clusters suggests a disorder-order transition between a ground and a sparsely populated higher energy state in the absence of ligands. Analysis of the faster, ps-ns motion of (15)N-(1)H bond vectors indicates an unusual nonlinear temperature-dependence for both ligation states. Intriguingly, while bile salt binding results in a more uniform response to temperature change throughout the protein, the temperature derivative of the generalized order parameter shows different responses to temperature increase for the two forms of the protein in the investigated temperature range. Analysis of both slow and fast motions in human I-BABP indicates largely different energy landscapes for the apo and holo states suggesting that optimization of binding interactions might be achieved by altering the dynamic behavior of specific segments in the protein.

Sodium selective ion channel formation in living cell membranes by polyamidoamine dendrimer.

Polyamidoamine (PAMAM) dendrimers are highly charged hyperbranched protein-like polymers that are known to interact with cell membranes. In order to disclose the mechanisms of dendrimer-membrane interaction, we monitored the effect of PAMAM generation five (G5) dendrimer on the membrane permeability of living neuronal cells followed by exploring the underlying structural changes with infrared-visible sum frequency vibrational spectroscopy (SVFS), small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). G5 dendrimers were demonstrated to irreversibly increase the membrane permeability of neurons that could be blocked in low-[Na(+)], but not in low-[Ca(2+)] media suggesting the formation of specific Na(+) permeable channels. SFVS measurements on silica supported DPPG-DPPC bilayers suggested G5-specific trans-polarization of the membrane. SAXS data and freeze-fracture TEM imaging of self-organized DPPC vesicle systems demonstrated disruption of DPPC vesicle layers by G5 through polar interactions between G5 terminal amino groups and the anionic head groups of DPPC. We propose a nanoscale mechanism by which G5 incorporates into the membrane through multiple polar interactions that disrupt proximate membrane bilayer and shape a unique hydrophilic Na(+) ion permeable channel around the dendrimer. In addition, we tested whether these artificial Na(+) channels can be exploited as antibiotic tools. We showed that G5 quickly arrest the growth of resistant bacterial strains below 10µg/ml concentration, while they show no detrimental effect on red blood cell viability, offering the chance for the development of new generation anti-resistant antibiotics.

Internal motions and exchange processes in human ileal bile acid binding protein as studied by backbone (15)N nuclear magnetic resonance spectroscopy.

Human ileal bile acid binding protein (I-BABP), a member of the family of intracellular lipid binding proteins, is thought to play a role in the enterohepatic circulation of bile salts. Previously, we have shown by stopped-flow fluorescence analysis that positive binding cooperativity exhibited by I-BABP in its interactions with glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two primary bile salts in humans, is related to a slow conformational change in the protein. In this study, we used backbone (15)N relaxation nuclear magnetic resonance (NMR) techniques to obtain residue-specific information about the internal dynamics of apo I-BABP and the doubly ligated I-BABP:GCA:GCDA complex on various time scales. According to our NMR data, bile salt binding is accompanied by a slight rigidification of the (15)N-(1)H bond vectors on the picosecond to nanosecond time scale, with most pronounced changes occurring in the C-D region. In contrast to the minor effects of ligation on fast motions, relaxation dispersion NMR experiments indicate a marked difference between the two protein states on the microsecond to millisecond time scale. In the apo form, an extensive network of conformational fluctuations is detected throughout segments of the EFGHIJ ß-strands and the C-D loop, which cease upon complexation. Our NMR data are in agreement with a conformational selection model we proposed earlier for I-BABP and support the hypothesis of an allosteric mechanism of ligand binding. According to the NMR measurements, the helical cap region may have a less crucial role in mediating ligand entry and release than what has been indicated for fatty acid binding proteins.