Ligand Binding Properties Research Articles

Structural and ligand-binding properties of fatty acid-binding proteins (pFABP4 and pFABP5) in the gentoo penguin

Structural and ligand-binding properties of fatty acid-binding proteins (pFABP4 and pFABP5) in the gentoo penguin

Biophysical and Biochemical Characterization of Avian Secretory Component Provides Structural Insights into the Evolution of the Polymeric Ig Receptor.

The polymeric Ig receptor (pIgR) transports polymeric Abs across epithelia to the mucosa, where proteolytic cleavage releases the ectodomain (secretory component [SC]) as an integral component of secretory Abs, or as an unliganded protein that can mediate interactions with bacteria. SC is conserved among vertebrates, but domain organization is variable: mammalian SC has five domains (D1-D5), whereas avian, amphibian, and reptilian SC lack the D2 domain, and fish SC lacks domains D2-D4. In this study, we used double electron-electron resonance spectroscopy and surface plasmon resonance binding studies to characterize the structure, dynamics, and ligand binding properties of avian SC, avian SC domain variants, and a human SC (hSC) variant lacking the D2 domain. These experiments demonstrated that, unlike hSC, which adopts a compact or "closed" domain arrangement, unliganded avian SC is flexible and exists in both closed and open states, suggesting that the mammalian SC D2 domain stabilizes the closed conformation observed for hSC D1-D5. Experiments also demonstrated that avian and mammalian pIgR share related, but distinct, mechanisms of ligand binding. Together, our data reveal differences in the molecular recognition mechanisms associated with evolutionary changes in the pIgR protein.

New palladium(II) hydrazone complexes: Synthesis, structure and biological evaluation

Two new palladium(II) complexes of 4-hydoxy-benzoic acid (5-bromo-2-hydroxy-benzylidene)-hydrazide (H2L) (1) with triphenylphosphine and triphenylarsine as coligand have been synthesized and characterized by the aid of various spectral techniques. The structure of the ligand and complexes was confirmed by single crystal X-ray diffraction studies. The hydrazone ligand acts as a tridendate ligand with ONO as the donor sites and is preferably found in the enol form in all the complexes. The structural analysis of 2 and 3 confirms the square planar geometry of the two complexes. The DNA binding of these complexes and ligand calf thymus DNA (CT-DNA) was investigated by using various methods, which revealed that the compounds interacted with CT-DNA through intercalation. Binding properties of the free ligand and its complexes with bovine serum albumin (BSA) protein have been investigated using UV–visible and fluorescence spectroscopic methods which indicated the stronger binding nature of the palladium complexes to BSA than the free hydrazone ligand. In addition, concentration dependent free radical scavenging potential of all the synthesized compounds (1–3) was also carried out under in vitro conditions. Further, the in vitro cytotoxicity of the compounds was examined on a HeLa and MCF-7 cell lines, which revealed that complex 2 exhibited a superior cytotoxicity than complex 3 and ligand 1.

Characterization of Kinetic Binding Properties of Unlabeled Ligands via a Preincubation Endpoint Binding Approach

The dissociation rates of unlabeled drugs have been well studied by kinetic binding analyses. Since kinetic assays are laborious, we developed a simple method to determine the kinetic binding parameters of unlabeled competitors by a preincubation endpoint assay. The probe binding after preincubation of a competitor can be described by a single equation as a function of time. Simulations using the equation revealed the degree of IC50 change induced by preincubation of a competitor depended on the dissociation rate koff of the competitor but not on the association rate kon. To validate the model, an in vitro binding assay was performed using a smoothened receptor (SMO) and [3H]TAK-441, a SMO antagonist. The equilibrium dissociation constants (KI) and koff of SMO antagonists determined by globally fitting the model to the concentration–response curves obtained with and without 24 h preincubation correlated well with those determined by other methods. This approach could be useful for early-stage optimization of drug candidates by enabling determination of binding kinetics in a high-throughput manner because it does not require kinetic measurements, an intermediate washout step during the reaction, or prior determination of competitors’ KI values.

Molecular and functional characterization of Toll-like receptor (Tlr)1 and Tlr2 in common carp (Cyprinus carpio).

Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs.

Four C1q domain-containing proteins involved in the innate immune response in Hyriopsis cumingii.

C1q is a key subcomponent of the complement C1 complex. This subcomponent contains a globular C1q (gC1q) domain with remarkable ligand binding properties. C1q domain-containing (C1qDC) proteins are composed of all proteins with a gC1q domain. C1qDC proteins exist in many invertebrates and recognize non-self-ligands. In our study, four C1qDC genes, namely, HcC1qDC1-HcC1qDC4, were identified from Hyriopsis cumingii. HcC1qDC1-HcC1qDC4 encode a protein of 224, 204, 305, and 332 amino acids, respectively. All C1qDC proteins consist of a gC1q domain at the C terminal. In addition to the gC1q domain, a coiled-coil region is found in HcC1qDC4. Multiple alignments and phylogenetic tree analysis revealed that the C1qDC proteins highly differ from one another. Tissue distribution analysis demonstrated that HcC1qDC1-HcC1qDC4 are widely distributed in hemocytes, hepatopancreas, gills, mantle, and foot. These C1qDC genes are regulated by bacteria to varying degrees. These recombinant HcC1qDC proteins exhibit a binding activity against different bacterial species. Our results may suggest the roles of HcC1qDC genes in anti-bacterial immune defense.

Type II Turn of Receptor-bound Salmon Calcitonin Revealed by X-ray Crystallography

Calcitonin is a peptide hormone consisting of 32 amino acid residues and the calcitonin receptor is a Class B G protein-coupled receptor (GPCR). The crystal structure of the human calcitonin receptor ectodomain (CTR ECD) in complex with a truncated analogue of salmon calcitonin ([BrPhe(22)]sCT(8-32)) has been determined to 2.1-Å resolution. Parallel analysis of a series of peptide ligands showed that the rank order of binding of the CTR ECD is identical to the rank order of binding of the full-length CTR, confirming the structural integrity and relevance of the isolated CTR ECD. The structure of the CTR ECD is similar to other Class B GPCRs and the ligand binding site is similar to the binding site of the homologous receptors for the calcitonin gene-related peptide (CGRP) and adrenomedulin (AM) recently published (Booe, J. M., Walker, C. S., Barwell, J., Kuteyi, G., Simms, J., Jamaluddin, M. A., Warner, M. L., Bill, R. M., Harris, P. W., Brimble, M. A., Poyner, D. R., Hay, D. L., and Pioszak, A. A. (2015) Mol. Cell 58, 1040-1052). Interestingly the receptor-bound structure of the ligand [BrPhe(22)]sCT(8-32) differs from the receptor-bound structure of the homologous ligands CGRP and AM. They all adopt an extended conformation followed by a C-terminal β turn, however, [BrPhe(22)]sCT(8-32) adopts a type II turn (Gly(28)-Thr(31)), whereas CGRP and AM adopt type I turns. Our results suggest that a type II turn is the preferred conformation of calcitonin, whereas a type I turn is the preferred conformation of peptides that require RAMPs; CGRP, AM, and amylin. In addition the structure provides a detailed molecular explanation and hypothesis regarding ligand binding properties of CTR and the amylin receptors.

Synthesis, characterization and biological studies on NiII and CuII complexes of two novel α,β-unsaturated 1,3-diketones related to curcuminoids

Microwave assisted synthesis of two novel α,β-unsaturated 1,3-diketones, their mononuclear NiII and CuII complexes, characterization, results on DNA binding activities, short term in vitro and in vivo antitumor activities were reported. The ligands are structurally related to curcuminoids and exhibited significant antitumor activity against Dalton’s lymphoma ascites cells. The ligands were found to be existing in their enolic form and metal complexes have 1:2 metal ligand stoichiometry evidenced by electronic, IR, 1H NMR, 13C NMR and FAB mass spectral data. The DNA binding properties of ligand and metal complexes were studied by absorption titrations and by viscosity measurements with calf thymus DNA. The intrinsic binding constants of copper(II) complexes are slightly higher than complexes of same metals reported in the literature. Interaction of copper(II) complexes were found to be higher than that of nickel(II) complexes and free ligands. The copper(II) complexes of the ligands were found to be more effective towards Dalton’s lymphoma ascites cells in both in vitro and in vivo studies.

Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. The studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.

Crystal structures of two monomeric triosephosphate isomerase variants identified via a directed-evolution protocol selecting for L-arabinose isomerase activity.

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using an Escherichia coli L-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of the Escherichia coli selection strain in medium supplemented with 40 mM L-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

Evolution of host-defense function of C-reactive protein from horseshoe crab to humans

Abstract C-reactive protein (CRP) has been conserved throughout evolution. Human native CRP exhibits calcium-dependent binding specificity for phosphocholine. Human CRP in its non-native structure expresses the capability to bind to deposited and conformationally-altered proteins and which can be achieved by several means including treatment of CRP with acidic pH. The ligand-binding property of human CRP in its non-native structure has implications for toxic and inflammatory conditions and favors the conservation of CRP throughout evolution. It is not known, however, whether CRP from invertebrates exhibits structure-based ligand-binding properties similar to that of human CRP. The aim of this study was to investigate the ligand-binding properties of CRP from the American horseshoe crab Limulus polyphemus. We used oxidized low-density lipoprotein (ox-LDL) immobilized on microtiter plates as a model for deposited and conformationally-altered proteins. We found that Limulus CRP binds to ox-LDL at physiological pH, in contrast to human CRP which requires acidic pH to do so. The binding of Limulus CRP to ox-LDL occurred even in the absence of calcium, suggesting that the binding was not mediated through exposed phosphocholine molecules, if any, on ox-LDL. We conclude that the host-defense function of CRP evolved with the development of the immune system to expose a ligand-binding specificity only when needed, that is, an inflammatory microenvironment would have to be sensed by CRP and that CRP would change its structure to execute its function. Limulus CRP also provides us with a tool to investigate the structure-function relationships of human CRP in animal models of inflammation.

The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors

Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [3H]granisetron (Ki = 6.76 μM) and G-FL (Ki = 4.90 μM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 μM, and competed with G-FL with a Ki of 7.94 μM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 μM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects.

Characterization and functional analysis of a recombinant tau class glutathione transferase GmGSTU2-2 from Glycine max

The plant tau class glutathione transferases (GSTs) perform diverse catalytic as well as non-catalytic roles in detoxification of xenobiotics, prevention of oxidative damage and endogenous metabolism. In the present work, the tau class isoenzyme GSTU2-2 from Glycine max (GmGSTU2-2) was characterized. Gene expression analysis of GmGSTU2 suggested a highly specific and selective induction pattern to osmotic stresses, indicating that gene expression is controlled by a specific mechanism. Purified, recombinant GmGSTU2-2 was shown to exhibit wide-range specificity towards xenobiotic compounds and ligand-binding properties, suggesting that the isoenzyme could provide catalytic flexibility in numerous metabolic conditions. Homology modeling and phylogenetic analysis suggested that the catalytic and ligand binding sites of GmGSTU2-2 are well conserved compared to other tau class GSTs. Structural analysis identified key amino acid residues in the hydrophobic binding site and provided insights into the substrate specificity of this enzyme. The results established that GmGSTU2-2 participates in a broad network of catalytic and regulatory functions involved in the plant stress response.

Insulin-Like Growth Factor-II/Cation-Independent Mannose 6-Phosphate Receptor in Neurodegenerative Diseases.

The insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6P) receptor is a multifunctional single transmembrane glycoprotein. Recent studies have advanced our understanding of the structure, ligand-binding properties, and trafficking of the IGF-II/M6P receptor. This receptor has been implicated in a variety of important cellular processes including growth and development, clearance of IGF-II, proteolytic activation of enzymes, and growth factor precursors, in addition to its well-known role in the delivery of lysosomal enzymes. The IGF-II/M6P receptor, distributed widely in the central nervous system, has additional roles in mediating neurotransmitter release and memory enhancement/consolidation, possibly through activating IGF-II-related intracellular signaling pathways. Recent studies suggest that overexpression of the IGF-II/M6P receptor may have an important role in regulating the levels of transcripts and proteins involved in the development of Alzheimer's disease (AD)-the prevalent cause of dementia affecting the elderly population in our society. It is reported that IGF-II/M6P receptor overexpression can increase the levels/processing of amyloid precursor protein leading to the generation of β-amyloid peptide, which is associated with degeneration of neurons and subsequent development of AD pathology. Given the significance of the receptor in mediating the transport and functioning of the lysosomal enzymes, it is being considered for therapeutic delivery of enzymes to the lysosomes to treat lysosomal storage disorders. Notwithstanding these results, additional studies are required to validate and fully characterize the function of the IGF-II/M6P receptor in the normal brain and its involvement in various neurodegenerative disorders including AD. It is also critical to understand the interaction between the IGF-II/M6P receptor and lysosomal enzymes in neurodegenerative processes, which may shed some light on developing approaches to detect and prevent neurodegeneration through the dysfunction of the receptor and the endosomal-lysosomal system.

Facing the challenge of a functional characterization of toll-like receptor (TLR)1 and TLR2 in common carp

Toll-like receptors are a family of germline-encoded pattern recognition receptors which activate rapid inflammatory responses upon detection of their cognate ligands. In mammals, TLR2 can form a heterodimer with TLR1, 6, or 10, and recognize lipoproteins/lipopeptides from Gram-positive bacteria. Of these TLRs, only tlr1 and tlr2 are present in fish genomes. In the carp genome, a single full-length tlr1 gene could be identified, whereas two functional genes for carp tlr2 exist. High expression of tlr1 and both tlr2 genes was found in immune organs and leukocytes of both myeloid and lymphoid origin. Of the two tlr2 genes, tlr2a was always higher expressed than tlr2b. Three-dimensional modelling of the carp Tlr1 and Tlr2 proteins appear to confirm the ability to form a heterodimer, with a pocket that can accommodate the tri-acylated lipopeptide ligand Pam3CSK4, similar to the heterodimer structure of human TLR1-TLR2 on which the model was built. In cell lines of human as well as fish origin transfected to overexpress the carp Tlr proteins, co-localization of carp Tlr1 and Tlr2 could be revealed with confocal microscopy. We were unable to confirm Tlr localization to the cell surface, the expected sub-cellular localization of Tlr1 and Tlr2. Further experimental evidence would be needed to unequivocally prove molecular interaction between the two Tlr proteins. In previous work, we have described ligand-specific activation of carp Tlr2 overexpressed in human cells (HEK), via measurement of increased phosphorylation levels of the MAP kinase p38 by Western blot. Instead of this semi-quantitative method, here we used a read-out system based on NF-κB activation and subsequent measurement of luminescence. This quantitative method could not confirm our initial ligand binding studies, not using human cell lines (HEK, HeLa), nor using a fish cell line (EPC). We will discuss possible explanations for the unsuccessful ligand-specific activation of NF-κB after overexpression of Tlr1 and/or Tlr2 in human, but also fish cell lines, to propose alternative future strategies for studying ligand binding properties of fish Tlrs.

Ligand-binding properties of three odorant-binding proteins of the diamondback moth Plutella xylostella

Strategies for insect population control are currently targeting chemical communication at the molecular level. The diamondback moth Plutella xylostella represents one of the most serious pests in agriculture, however detailed information on the proteins mediating olfaction in this species is still poor. This species is endowed with a repertoire of a large number of olfactory receptors and odorant binding proteins (OBPs). As a contribution to map the specificities of these chemical sensors in the moth and eventually unravel the complexity of chemodetection, we have measured the affinities of three selected OBPs to a series of potential odorants. Three proteins are highly divergent in their amino acid sequences and show markedly different expression profiles. In fact, PxylOBP3 is exclusively expressed in the antennae of both sexes, PxylOBP9 is male specific and present only in antennae and reproductive organs, while PxylOBP19, an unusual OBP with nine cysteines, is ubiquitously present in all the organs examined. Such expression pattern suggests that the last two proteins may be involved in non-chemosensory functions. Despite such differences, the three OBPs exhibit similar binding spectra, together with high selectivity. Among the 26 natural compounds tested, only two proved to be good ligands, retinol and coniferyl aldehyde. This second compound is particularly interesting being part of the chemical pathway leading to regeneration of lignin, one of the defense strategies of the plant against insect attack, and might find applications as a repellent for P. xylostella and other pests.

Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering.

Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication–degeneration–complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor–ligand interactions of NRs following WGDs.

Dopaminergic receptor–ligand binding assays based on molecularly imprinted polymers on quartz crystal microbalance sensors

Molecularly imprinted polymers (MIPs) have been successfully applied as selective materials for assessing the binding activity of agonist and antagonist of dopamine D1 receptor (D1R) by using quartz crystal microbalance (QCM). In this study, D1R derived from rat hypothalamus was used as a template and thus self-organized on stamps. Those were pressed into an oligomer film consisting of acrylic acid: N-vinylpyrrolidone: N,N′-(1,2-dihydroxyethylene) bis-acrylamide in a ratio of 2:3:12 spin coated onto a dual electrode QCM. Such we obtained one D1R-MIP-QCM electrode, whereas the other electrode carried the non-imprinted control polymer (NIP) that had remained untreated. Successful imprinting of D1R was confirmed by AFM. The polymer can re-incorporate D1R leading to frequency responses of 100–1200Hz in a concentration range of 5.9–47.2µM. In a further step such frequency changes proved inherently useful for examining the binding properties of test ligands to D1R. The resulting mass-sensitive measurements revealed Kd of dopamine∙HCl, haloperidol, and (+)-SCH23390 at 0.874, 25.6, and 0.004nM, respectively. These results correlate well with the values determined in radio ligand binding assays. Our experiments revealed that D1R-MIP sensors are useful for estimating the strength of ligand binding to the active single site. Therefore, we have developed a biomimetic surface imprinting strategy for QCM studies of D1R-ligand binding and presented a new method to ligand binding assay for D1R.

Regulation of Mitochondrial Signaling and Quality Control by the 18KDA Translocator Protein (TSPO)

The 18-kDa translocator protein (TSPO) is localized to the outer mitochondrial membrane (OMM) where it is found in complex with the voltage dependent anion channel (VDAC). TSPO expression is elevated in cancer and inflammatory conditions and it is well known for its ligand binding properties and application in PET imaging as a marker of neuroinflammation. TSPO is implicated in multiple mitochondrial functions including steroidogenesis, apoptosis and ATP production although precise molecular mechanisms remain to be elucidated. We recently demonstrated that increased TSPO expression limits mitochondrial ubiquitination and mitophagy through a reactive oxygen species (ROS) -dependent process, which leads to changes in mitochondrial morphology and biomass.Here we have corroborated this pathway by establishing an interplay with the protein kinase A (PKA) signaling complex, which is recruited to mitochondria by TSPO via acyl-CoA binding domain containing 3 (ACBD3). Using targeted luminescent protein chimeras, we show that the combined accumulation of TSPO and PKA alters subcellular Ca2+ dynamics and metabolism, which we propose arises from increased VDAC phosphorylation and subsequent changes in channel activity. Of note, this pathway is activated under glutamate-induced cell stress in the neuronal cell line, SH-SY5Y.This work highlights an important role for the OMM in fine-tuning mitochondrial signaling and mitophagy. As an inducible factor, TSPO represents a critical physiological element that facilitates mitochondrial adaptation to altered cellular metabolic requirements under stress conditions.

Thermal Stability of Human Serum Albumin: the Dependence on the Protein Concentration, Scan Rate, and the Presence of Fatty Acids and Low-Weight Molecular Ligands

Human serum albumin (HSA) is the most abundant protein in human plasma with ascribed ligand binding and transport properties. Design of this molecule provides a variety of binding sites for many physiological important ligands, including a wide variety of drugs. In this work, the dependence of the thermal denaturation of HSA on the protein concentration, scan rate, and the presence of fatty acids and low-molecular weight drugs (warfarin, ibuprofen, hypericin, emodin) have been thoroughly investigated by means of differential scanning calorimetry (DSC). Our results show that the presence of fatty acids in HSA significantly stabilizes the structure of this protein. Further, DSC curves exhibit a significant dependence on the protein concentration in both fatty acid containing and fatty acids free HSA. From the linear dependence of 1/Tm on albumin concentration follows that HSA tends to form dimers during denaturation process. The obtained DSC data show only a slight increase of Tm and ΔHcal with a scan rate increase. Interactions of the studied low-molecular weight drugs with HSA lead to a significant enhancement of the protein thermal stability (higher temperature and enthalpy of the denaturation in comparison with those thermodynamic parameters determined for HSA alone). However, in the presence of emodin at high concentration ratios (emodin:HSA>10:1), a decrease of the stabilization effect is observed. This is probably caused by the ability of emodin to bind both folded and unfolded states of HSA.AcknowledgementsThis work has been supported by the contract APVV-0242-11, the project CELIM (316310) funded by 7 FP EU, and by the EU Structural Funds (ITMS 26240120040 and ITMS 26220220107(50%)).