The Multienzyme Complex Nature of Dehydroepiandrosterone Sulfate Biosynthesis.

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein-protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes.

The effect of membrane composition on the interaction between human CYP51 and its flavonoid inhibitor - luteolin 7,3'-disulfate.

Cytochromes P450 (CYP) are a family of membrane proteins involved in the production of endogenous molecules and the metabolism of xenobiotics. It is well-known that the composition of the membrane can influence the activity and orientation of CYP proteins. However, little is known about how membrane composition affects the ligand binding properties of CYP. In this study, we utilized surface plasmon resonance and fluorescence lifetime analysis to examine the impact of membrane micro-environment composition on the interaction between human microsomal CYP51 (CYP51A1) and its inhibitor, luteolin 7,3'-disulphate (LDS). We observed that membranes containing cholesterol or sphingomyelin exhibited the lowest apparent equilibrium dissociation constant for the CYP51A1-LDS complex. Additionally, the tendency for relation between kinetic parameters of the CYP51A1-LDS complex and membrane viscosity and overall charge was observed. These findings suggest that the specific composition of the membrane, particularly the presence of cholesterol and sphingomyelin, plays a vital role in regulating the interaction between CYP enzymes and their ligands.

Proteomic Profiling of Mouse Brain Pyruvate Kinase Binding Proteins: A Hint for Moonlighting Functions of PKM1?

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein-protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.

Protein Interactome Profiling of Stable Molecular Complexes in Biomaterial Lysate.

Most proteins function as part of various complexes, forming via stable and dynamic protein-protein interactions (PPIs). The profiling of PPIs expands the fundamental knowledge about the structures, functions, and regulation patterns of protein complexes and intracellular molecular machineries. Protein interactomics aims at solving three main tasks: (1) identification of protein partners and parts of complex intracellular structures; (2) analysis of PPIs parameters (affinity, molecular-recognition specificity, kinetic rate constants, and thermodynamic-parameters determination); (3) the study of the functional role of novel PPIs. The purpose of this work is to update the current state and prospects of multi-omics approaches to profiling of proteins involved in the formation of stable complexes. Methodological paradigm includes a development of protein-extraction and -separation techniques from tissues or cellular lysates and subsequent identification of proteins using mass-spectrometry analysis. In addition, some aspects of authors' experimental platforms, based on high-performance size-exclusion chromatography, procedures of molecular fishing, and protein identification, as well as the possibilities of interactomic taxonomy of each protein, are discussed.

Structural insights into 3Fe-4S ferredoxins diversity in M. tuberculosis highlighted by a first redox complex with P450.

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis-Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high-affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context.

A global protein interactome ensures the maintenance of regulatory, signaling and structural processes in cells, but at the same time, aberrations in the repertoire of protein-protein interactions usually cause a disease onset. Many metabolic enzymes catalyze multistage transformation of cholesterol precursors in the cholesterol biosynthesis pathway. Cancer-associated deregulation of these enzymes through various molecular mechanisms results in pathological cholesterol accumulation (its precursors) which can be disease risk factors. This work is aimed at systematization and bioinformatic analysis of the available interactomics data on seventeen enzymes in the cholesterol pathway, encoded by HMGCR, MVK, PMVK, MVD, FDPS, FDFT1, SQLE, LSS, DHCR24, CYP51A1, TM7SF2, MSMO1, NSDHL, HSD17B7, EBP, SC5D, DHCR7 genes. The spectrum of 165 unique and 21 common protein partners that physically interact with target enzymes was selected from several interatomic resources. Among them there were 47 modifying proteins from different protein kinases/phosphatases and ubiquitin-protein ligases/deubiquitinases families. A literature search, enrichment and gene co-expression analysis showed that about a quarter of the identified protein partners was associated with cancer hallmarks and over-represented in cancer pathways. Our results allow to update the current fundamental view on protein-protein interactions and regulatory aspects of the cholesterol synthesis enzymes and annotate of their sub-interactomes in term of possible involvement in cancers that will contribute to prioritization of protein targets for future drug development.

Human Lanosterol 14-Alpha Demethylase (CYP51A1) Is a Putative Target for Natural Flavonoid Luteolin 7,3'-Disulfate.

Widespread pathologies such as atherosclerosis, metabolic syndrome and cancer are associated with dysregulation of sterol biosynthesis and metabolism. Cholesterol modulates the signaling pathways of neoplastic transformation and tumor progression. Lanosterol 14-alpha demethylase (cytochrome P450(51), CYP51A1) catalyzes one of the key steps in cholesterol biosynthesis. The fairly low somatic mutation frequency of CYP51A1, its druggability, as well as the possibility of interfering with cholesterol metabolism in cancer cells collectively suggest the clinical importance of CYP51A1. Here, we show that the natural flavonoid, luteolin 7,3'-disulfate, inhibits CYP51A1 activity. We also screened baicalein and luteolin, known to have antitumor activities and low toxicity, for their ability to interact with CYP51A1. The Kd values were estimated using both a surface plasmon resonance optical biosensor and spectral titration assays. Unexpectedly, in the enzymatic activity assays, only the water-soluble form of luteolin-luteolin 7,3'-disulfate-showed the ability to potently inhibit CYP51A1. Based on molecular docking, luteolin 7,3'-disulfate binding suggests blocking of the substrate access channel. However, an alternative site on the proximal surface where the redox partner binds cannot be excluded. Overall, flavonoids have the potential to inhibit the activity of human CYP51A1 and should be further explored for their cholesterol-lowering and anti-cancer activity.

A new insight into subinteractomes of functional antagonists: Thromboxane (CYP5A1) and prostacyclin (CYP8A1) synthases.

The current article aims to summarize all possible spectrum of protein-protein interactions for thromboxane A synthase (CYP5A1) and prostacyclin synthase (CYP8A1). These enzymes metabolize the same substrate (prostaglandin H2 ) and can participate in cardiovascular, inflammatory, immune processes, and apoptosis modulation, as well as significantly influence the risk of cancers. Binary protein-protein and multiprotein complexes are of great importance in enzyme-regulating and signal-transduction pathways. However, protein partners of CYP5A1 and CYP8A1 are not yet fully identified, although both synthases are considered as prospective drug targets. At least 36 novel protein partners of CYP5A1 and CYP8A1 were revealed from different tissue types using an approach based on affinity isolation and mass spectrometry. Enrichment analysis showed that these proteins have different molecular functions: folding (refolding), unfolded protein and chaperon binding, protein transport (export/import), posttranslational modification, protein domain-specific binding, antioxidant activity, and glutathione homeostasis. A significant part of them, belonging to molecular chaperones, were common partners for CYP5A1 and CYP8A1, while other proteins were unique with the tissue-dependent distribution. New aspects of CYP5A1 and CYP8A1 interactomics and hetero-complex formation with different protein partners, including cytochrome P450s are discussed.

A large-scale comparative analysis of affinity, thermodynamics and functional characteristics of interactions of twelve cytochrome P450 isoforms and their redox partners.

The aim of the present work was to establish the thermodynamic and functional differences in the protein-protein interactions between the components of the P450-dependent mitochondrial (mit) and microsomal (mic) monooxygenase systems using 12 different isoforms of cytochromes P450 and two redox partners, NADPH-dependent cytochrome P450 reductase (CPR) and adrenodoxin (Adx). Comparative analysis of the affinity, thermodynamics, enzymatic activity and the ability for one-electron reduction has been carried out. The study of protein-protein interactions to determine the equilibrium dissociation constants (Kd) was performed using surface plasmon resonance (SPR) biosensor Biacore 3000. We demonstrated that CPR and Adx interacted with both, micCYPs and mitCYPs, with different affinities (Kd values ranged from 0.01 to 2 µM). All complexes of microsomal (micCYP) and mitochondrial (mitCYP) cytochrome P450 with redox partners can be divided into three groups depending on the prevalent role of either enthalpy or entropy contribution. About 90% of CYP/redox partner complexes were entropy-driven, while the contribution of enthalpy and entropy differed significantly in case of mitCYP/Adx complexes. The CYP11A1/Adx complex was enthalpy-driven, while CYP11B1/Adx and CYP11B2/Adx complexes were entropy-driven. Thermodynamic discrimination of mitCYPs/Adx complexes is likely associated with the different functional impact of CYP11A1 and CYP11B. The exception was the enthalpy-entropy-driven (mixed type) CYP21A2/Adx complex. CPR and Adx were able to transfer the first electron to micCYPs while mitCYPs demonstrated high specificity to Adx. Productive catalysis for mitCYPs observed only in the presence of Adx/AdR pair, while in case of steroidogenic micCYPs (CYP17A1, CYP19A1, and CYP21A2) it was found either in the presence of a CPR or an Adx/AdR pair. From the evolutionary point of view, the type 1 electron transport system (mitCYPs, Adx and NADPH-dependent adrenodoxin reductase (AdR)) increased the specialization of protein-protein interactions (PPI) significantly, which was accompanied by an increase in the specificity of electron transfer. In contrast, the evolution of the type 2 electron transport system (micCYPs and CPR) led to an increase in versatility of PPI as demonstrated for steroidogenic microsomal cytochrome P450s. Our data enhance the current understanding of molecular recognition and summarize qualitative and thermodynamic characteristics of protein-protein interactions in the P450-dependent mitochondrial and microsomal monooxygenase systems.

Isatin, an endogenous nonpeptide biofactor: A review of its molecular targets, mechanisms of actions, and their biomedical implications.

Isatin (indole-2,3-dione) is an oxidized indole. It is widely distributed in mammalian tissues and body fluids, where isatin concentrations vary significantly from <0.1 to > 10 µM. Isatin output is increased under conditions of stress. Exogenously administered isatin is characterized by low toxicity, mutagenicity, and genotoxicity in vivo. Cytotoxic effects of isatin on various cell cultures are usually observed at concentrations exceeding 100 µM. Binding of [3 H]isatin to rat brain sections is consistent with its physiological concentrations. Proteomic analysis of mouse and rat brain isatin-binding proteins revealed about 90 individual proteins, which demonstrated significant interspecies differences (rat versus mouse). Certain evidence exist that redox state(s) and possibly other types of posttranslational modifications regulate affinity of target proteins to isatin. Recent data suggest that interacting with numerous intracellular isatin binding proteins, isatin can act as a regulator of complex protein networks in norm and pathology. Physiological concentrations of isatin in vitro inhibit monoamine oxidase B and natriuretic peptide receptor guanylate cyclase, higher (neuroprotective) concentrations (50-400 µM) cause apoptosis of various (including malignant tumor) cell lines and influence expression of certain apoptosis-related genes. Being administered in vivo, isatin exhibits various behavioral effects; it attenuates manifestations of MPTP-induced parkinsonism and tumor growth in experimental animal models. © 2017 BioFactors, 44(2):95-108, 2018.

New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1ß expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.

Computer-aided design of aptamers for cytochrome p450.

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M.

Amyloid-binding proteins: affinity-based separation, proteomic identification, and optical biosensor validation.

The amyloid-beta peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-beta accumulates inside cells, intracellular brain amyloid-beta-binding proteins remain poorly characterized. Here we describe a protocol for affinity-based profiling of amyloid-beta-binding proteins of rat brain, their proteomic identification and validation by a surface plasmon resonance (SPR)-based analysis. It includes: (a) SPR-based selection of immobilization conditions for beta-amyloid coupling and choice of appropriate resin for preparation of an affinity sorbent; (b) immobilization of beta-amyloid on the selected resin;

Protein interactomics based on direct molecular fishing on paramagnetic particles: practical realization and further SPR validation.

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity-based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC-MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR-based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 µM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein-protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.

The effects of endogenous non-peptide molecule isatin and hydrogen peroxide on proteomic profiling of rat brain amyloid-ß binding proteins: relevance to Alzheimer's disease?

The amyloid-ß peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-ß accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-ß binding proteins and 0.1 mM isatin decreased the number of identified amyloid-ß binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-ß binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-ß oligomers described in the literature for some isatin derivatives.

Highly sensitive detection of human cardiac myoglobin using a reverse sandwich immunoassay with a gold nanoparticle-enhanced surface plasmon resonance biosensor.

A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10 pM (approx. 0.18 ng mL(-1)), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.

Protein interactomics based on direct molecular fishing on paramagnetic particles: experimental simulation and SPR validation.

We describe an experimental approach for direct molecular fishing of prey protein on the surface of two types of paramagnetic particles (PMP) having different size and composition. Human microsomal cytochrome b(5) (b(5)) and its known partner human cytochrome P450 3A5 (CYP3A5) were used as bait and prey proteins, respectively. For assessing the level of unspecific binding of background proteins, α-fetoprotein (aFP) was used. SPR measurements were applied for quantitative analysis of trapped proteins (CYP3A5 and aFP) after fishing on PMP. It was shown that the described approach of molecular fishing on micro-PMP provides enough prey proteins for LC-MS/MS identification and SPR validation, so this approach can be used for discovery of new protein-protein interactions in the framework of Human Proteome Project.

FMN binding site of yeast NADPH-cytochrome P450 reductase exposed at the surface is highly specific.

NADPH-cytochrome P450 reductase (CPR) transfers two reducing equivalents derived from NADPH via FAD and FMN to microsomal P450 monooxygenases in one-electron transfer steps. The crystal structure of yeast CPR (yCPR) contains a surface-exposed FMN binding site (FMN2 site) at the interface of the FMN binding and connecting domains, in addition to the single buried site that has been observed in rat CPR. This finding provides a testable hypothesis of how intramolecular (between FAD and FMN) and intermolecular (between FMN and P450) electron transfer may occur in CPR. To verify that occupancy of the FMN2 site is not an artifact of crystallization, a surface plasmon resonance (SPR) biosensor technique has been applied to probe the selectivity of this site under functional conditions. A series of kinetic and equilibrium binding experiments involving yCPR immobilized on different sensor chip surfaces was performed using FMN and FAD, as well as FMN-derived compounds, including riboflavin, dimethylalloxazine, and alloxazine, and other molecules that resemble the planar isoalloxazine ring structure. Only FMN and FAD showed stoichiometric binding responses. Binding affinity for FMN was in the submicromolar range, 30 times higher than that for FAD. Association kinetic rates for the yCPR/FMN complex were up to 60-fold higher than for the yCPR/FAD complex. Taken together, these data indicate that (i) the surface-exposed site in yCPR is highly selective toward binding flavins, (ii) binding of FMN in this site is notably favored, and finally, (iii) both the phosphate group and the isoalloxazine ring of FMN are essential for binding.

Electrochemical nanobiosensor for express diagnosis of acute myocardial infarction in undiluted plasma.

The myocardial infarction biomarker myoglobin was quantified at the biological level in undiluted plasma using developed electrochemical nanosensors with immobilized anti-myoglobin. Method for cardiac myoglobin detection is based on direct electron transfer between Fe(III)-heme and electrode surface modified with gold nanoparticles/didodecyldimethylammonium bromide (DDAB/Au) and antibodies. The procedure of myoglobin detection was optimized (pH, incubation times and characteristics of electrodes) to express determination of the marker in serum or plasma. Plasma of healthy donors and patients with acute myocardial infarction (AMI) was analyzed using electrochemical immunosensors and RAMP immunoassay. Square wave voltammetry cathodic peak of cardiac myoglobin reduction was found to be proportional to myoglobin quantity in plasma as determined by RAMP. The method proposed does not require signal enhancement or amplification; nor does it require labeled secondary antibodies. Immunosensor has a detection limit of 10 ng/ml (0.56 nM) and a broad range of working concentrations (10-1780 ng/ml; 0.56-100 nM). The whole procedure takes 30 min and can be used for express diagnosis of acute myocardial infarction.

Isatin-binding proteins of rat and mouse brain: proteomic identification and optical biosensor validation.

Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X-100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin-binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5-aminocarproyl-isatin, and 5-aminoisatin as the affinity ligands. The K(d) values (of 0.1-20 microM) obtained during the optical biosensor experiments were consistent with the range of K(d) values recently reported for [(3)H]isatin binding to brain sections. Although the number of isatin-binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one-third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.