Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Generation of aldehyde-derived protein modifications in ethanol-exposed heart

Background: Although excessive ethanol consumption is known to lead to a variety of adverse effects in the heart, the molecular mechanisms of such effects have remained poorly defined. We hypothesized that posttranslational covalent binding of reactive molecular species to proteins occurs in the heart in response to acute ethanol exposure. Methods: The generation of protein adducts with several aldehydic species was examined by using monospecific antibodies against adducts with malondialdehyde (MDA), acetaldehyde (AA), MDA-AA hybrids, and hydroxyethyl radicals. Specimens of heart tissue were obtained from rats after intraperitoneal injections with alcohol (75 mmol/kg body weight) with or without pretreatment with cyanamide (0.05 mmol/kg body weight), an aldehyde dehydrogenase inhibitor. Results: The amounts of MDA and unreduced AA adducts were found to be significantly increased in the heart of the rats treated with ethanol, cyanamide, or both, whereas no other adducts were detected in statistically significant quantities. Immunohistochemical studies for characterization of adduct distribution revealed sarcolemmal adducts of both MDA and AA in the rats treated with ethanol and cyanamide in addition to intracellular adducts, which were also present in the group treated with ethanol alone. Conclusions: These findings support the role of enhanced lipid peroxidation and the generation of protein-aldehyde condensates in vivo as a result of excessive ethanol intake. These findings may have implications in the molecular mechanisms of cardiac dysfunction in alcoholics.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.

An image processing application for quantification of protein aggregates in Caenorhabditis elegans

Protein aggregation became a widely accepted marker of many polyQ disorders, including Machado-Joseph disease (MJD), and is often used as readout for disease progression and development of therapeutic strategies. The lack of good platforms to rapidly quantify protein aggregates in a wide range of disease animal models prompted us to generate a novel image processing application that automatically identifies and quantifies the aggregates in a standardized and operator-independent manner. We propose here a novel image processing tool to quantify the protein aggregates in a Caenorhabditis elegans (C. elegans) model of MJD. Confocal mi-croscopy images were obtained from animals of different genetic conditions. The image processing application was developed using MeVisLab as a platform to pro-cess, analyse and visualize the images obtained from those animals. All segmenta-tion algorithms were based on intensity pixel levels.The quantification of area or numbers of aggregates per total body area, as well as the number of aggregates per animal were shown to be reliable and reproducible measures of protein aggrega-tion in C. elegans. The results obtained were consistent with the levels of aggrega-tion observed in the images. In conclusion, this novel imaging processing applica-tion allows the non-biased, reliable and high throughput quantification of protein aggregates in a C. elegans model of MJD, which may contribute to a significant improvement on the prognosis of treatment effectiveness for this group of disor-ders

Automatic segmentation and 3D feature extraction of protein aggregates in Caenorhabditis Elegans

In the last years, it has become increasingly clear that neurodegenerative diseases involve protein aggregation, a process often used as disease progression readout and to develop therapeutic strategies. This work presents an image processing tool to automatic segment, classify and quantify these aggregates and the whole 3D body of the nematode Caenorhabditis Elegans. A total of 150 data set images, containing different slices, were captured with a confocal microscope from animals of distinct genetic conditions. Because of the animals’ transparency, most of the slices pixels appeared dark, hampering their body volume direct reconstruction. Therefore, for each data set, all slices were stacked in one single 2D image in order to determine a volume approximation. The gradient of this image was input to an anisotropic diffusion algorithm that uses the Tukey’s biweight as edge-stopping function. The image histogram median of this outcome was used to dynamically determine a thresholding level, which allows the determination of a smoothed exterior contour of the worm and the medial axis of the worm body from thinning its skeleton. Based on this exterior contour diameter and the medial animal axis, random 3D points were then calculated to produce a volume mesh approximation. The protein aggregations were subsequently segmented based on an iso-value and blended with the resulting volume mesh. The results obtained were consistent with qualitative observations in literature, allowing non-biased, reliable and high throughput protein aggregates quantification. This may lead to a significant improvement on neurodegenerative diseases treatment planning and interventions prevention

Novel Scorpionate and Pyrazole Dioxovanadium Complexes, Catalysts for Carboxylation and Peroxidative Oxidation of Alkanes

The dioxovanadium(V) complexes [VO2(3,5-Me(2)Hpz)(3)][BF4] (1) (pz = pyrazolyl), [VO2{SO3C(pz)(3)}] (2), [VO2{HB(3,5-Me(2)pz)(3)}] (3) and [VO2{HC(pz)(3)}][BF4] (4), bearing pyrazole or scorpionate ligands, were obtained by reaction of triethyl vanadate [VO(OEt)(3)] with hydrotris(3,5-dimethyl-1-pyrazolyl)methane [HC(3,5-Me(2)pz)(3)] or 3,5-dimethylpyrazole (3,5-Me(2)Hpz; 1), lithium tris(1-pyrazolyl)methanesulfonate {Li[SO3C(pz)(3)], 2}, potassium hydrotris(3,5-dimethyl-1-pyrazolyl)borate {K[HB(3,5-Me(2)pz)(3)], 3} and hydrotris(1-pyrazolyl)methane [HC(pz)(3), 4], respectively. Treatment of [VO(OEt)(3)] with potassium hydrotris(1-pyrazolyl)borate {K[HB(pz)(3)]} led to the mixed eta(3)-tris(pyrazolyl)borate and eta(2)-bis(pyrazolyl)borate oxovanadium(IV) complex [VO{HB(pz)(3)}{H2B(pz)(2)}, 5]. The compounds were characterized by elemental analyses, IR, NMR and EPR spectroscopy, FAB and ESI mass spectrometry, cyclic voltammetry and, for 5, also by single crystal X-ray diffraction analysis. All complexes exhibit catalytic activity in the single-pot carboxylation [in trifluoroacetic acid/potassium peroxodisulfate (CF3COOH/K2S2O8)] of gaseous alkanes (methane and ethane) to carboxylic acids (yields up to 40%. TONs up to 157) and in the peroxidative oxidation [in water/acetonitrile (H2O/NCMe)] of liquid alkanes (cyclohexane and cyclopentane) to the corresponding alcohols and ketones (yields up to 24%, TONs up to 117), under mild conditions.

Extraction of hemoglobin with calixarenes and biocatalysis in organic media of the complex with pseudoactivity of peroxidase

The present work involves the use of p-tert-butylcalix[4,6,8]arene carboxylic acid derivatives ((t)Butyl[4,6,8]CH2COOH) for selective extraction of hemoglobin. All three calixarenes extracted hemoglobin into the organic phase, exhibiting extraction parameters higher than 0.90. Evaluation of the solvent accessible positively charged amino acid side chains of hemoglobin (PDB entry 1XZ2) revealed that there are 8 arginine, 44 lysine and 30 histidine residues on the protein surface which may be involved in the interactions with the calixarene molecules. The hemoglobin-(t)Butyl[6]CH2COOH complex had pseudoperoxidase activity which catalysed the oxidation of syringaldazine in the presence of hydrogen peroxide in organic medium containing chloroform. The effect of pH, protein and substrate concentrations on biocatalysis was investigated using the hemoglobin-(t)Butyl[6]CH2COOH complex. This complex exhibited the highest specific activity of 9.92 x 10(-2) U mg protein(-1) at an initial pH of 7.5 in organic medium. Apparent kinetic parameters (V'(max), K'(m), k'(cat) and k'(cat)/K'(m)) for the pseudoperoxidase activity were determined in organic media for different pH values from a Michaelis-Menten plot. Furthermore, the stability of the protein-calixarene complex was investigated for different initial pH values and half-life (t(1/2)) values were obtained in the range of 1.96 and 2.64 days. Hemoglobin-calixarene complex present in organic medium was recovered in fresh aqueous solutions at alkaline pH, with a recovery of pseudoperoxidase activity of over 100%. These results strongly suggest that the use of calixarene derivatives is an alternative technique for protein extraction and solubilisation in organic media for biocatalysis.

Bioconversion of D-glucose into D-glucosone by Glucose 2-oxidase from Coriolus versicolor at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose: oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of D-glucose at carbon 2 in the presence of molecular O(2) producing D-glucosone (2-keto-glucose and D-arabino-2-hexosulose) and H(2)O(2). It was used to convert D-glucose into D-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of D-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H(2)O(2) acted as inhibitor for this reaction. The rate of bioconversion of D-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO(2) at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55 degrees C) and pH (5.0) of D-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E(a)) was 32.08 kJmol(-1) and kinetic parameters (V(max), K(m), K(cat) and K(cat)/K(m)) for this bioconversion were 8.8 Umg(-1) protein, 2.95 mM, 30.81 s(-1) and 10,444.06 s(-1)M(-1), respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of D-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.

Bioconversion of D-glucose into D-glucosone by immobilized glucose 2-oxidase from Coriolus versicolorat moderate pressures

The immobilized glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor was used to convert D-glucose into D-glucosone at moderate pressures, up to 150 bar, with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, different forms of immobilized biocatalysts, glucose concentration, pH, temperature and the presence of catalase. Glucose 2-oxidase (GOX2) was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. Purified enzyme and catalase were immobilized into a polyethersulfone (PES) membrane in the presence of glutaraldehyde and gelatin. Enhancement of the bioconversion of D-glucose was done by the pressure since an increase in the pressure with compressed air increases the conversion rates. The optimum temperature and pH for bioconversion of D-glucose were found to be 62 degrees C and pH 6.0, respectively and the activation energy (E(a)) was 28.01 kJ mol(-1). The apparent kinetic constants (V(max)' K(m)', K(cat)' and K(cat)/K(m)') for this bioconversion were 2.27 U mg(-1) protein, 11.15 mM, 8.33 s(-1) and 747.38 s(-1) M(-1), respectively. The immobilized biomass of C. versicolor as well as crude extract containing GOX2 activity were also useful for bioconversion of D-glucose at 65 bar with a yield of 69.9 +/- 3.8% and 91.3 +/- 1.2%, respectively. The immobilized enzyme was apparently stable for several months without any significant loss of enzyme activity. On the other hand, this immobilized enzyme was also stable at moderate pressures, since such pressures did not affect significantly the enzyme activity. (C) 2010 Elsevier Ltd. All rights reserved.

Complexes of copper(II) with 3-(ortho-substituted phenylhydrazo)pentane-2,4-diones: syntheses,properties and catalytic activity for cyclohexane oxidation

Reactions of copper(II) with 3-phenylhydrazopentane-2,4-diones X-2-C6H4-NHN = C{C(= O)CH3}(2) bearing a substituent in the ortho-position [X = OH (H2L1) 1, AsO3H2 (H3L2) 2, Cl (HL3) 3, SO3H (H2L4) 4, COOCH3 (HL5) 5, COOH (H2L6) 6, NO2 (HL7) 7 or H (HL8) 8] lead to a variety of complexes including the monomeric [CuL4(H2O)(2)]center dot H2O 10, [CuL4(H2O)(2)] 11 and [Cu(HL4)(2)(H2O)(4)] 12, the dimeric [Cu-2(H2O)(2)(mu-HL2)(2)] 9 and the polymeric [Cu(mu-L-6)](n)] 13 ones, often bearing two fused six-membered metallacycles. Complexes 10-12 can interconvert, depending on pH and temperature, whereas the Cu(II) reactions with 4 in the presence of cyanoguanidine or imidazole (im) afford the monomeric compound [Cu(H2O)(4){NCNC(NH2)(2)}(2)](HL4)(2)center dot 6H(2)O 14 and the heteroligand polymer [Cu(mu-L-4)(im)](n) 15, respectively. The compounds were characterized by single crystal X-ray diffraction (complexes), electrochemical and thermogravimetric studies, as well as elemental analysis, IR, H-1 and C-13 NMR spectroscopies (diones) and ESI-MS. The effects of the substituents in 1-8 on the HOMO-LUMO gap and the relative stability of the model compounds [Cu(OH)(L-8)(H2O)]center dot H2O, [Cu(L-1)(H2O)(2)]center dot H2O and [Cu(L-4)(H2O)(2)]center dot H2O are discussed on the basis of DFT calculations that show the stabilization follows the order: two fused 6-membered > two fused 6-membered/5-membered > one 6-membered metallacycles. Complexes 9, 10, 12 and 13 act as catalyst precursors for the peroxidative oxidation (with H2O2) of cyclohexane to cyclohexanol and cyclohexanone, in MeCN/H2O (total yields of ca. 20% with TONs up to 566), under mild conditions.

Synthesis and structural characterization of iron complexes with 2,2,2-tris(1-pyrazolyl)ethanol ligands: Application in the peroxidative oxidation of cyclohexane under mild conditions

The reactions of FeCl2 center dot 2H(2)O and 2,2,2-tris(1-pyrazolyl) ethanol HOCH2C(pz)(3) (1) (pz = pyrazolyl) afford [Fe{HOCH2C(pz)(3)}(2)][FeCl4]Cl (2), [Fe{HOCH2C(pz)(3)}(2)](2)[Fe2OCl6](Cl)(2)center dot 4H(2)O (3 center dot 4H(2)O), [Fe{HOCH2C(pz)(3)}(2)] [FeCl{HOCH2C(pz)(3)}(H2O)(2)](2)(Cl)(4) (4) or [Fe{HOCH2C(pz)(3)}(2)]Cl-2 (5), depending on the experimental conditions. Compounds 1-5 were isolated as air-stable crystalline solids and fully characterized, including (1-4) by single-crystal X-ray diffraction analyses. The latter technique revealed strong intermolecular H-bonds involving the OH group of the scorpionate 2 and 3 giving rise to 1D chains which, in 3, are further expanded to a 2D network with intercalated infinite and almost plane chains of H-interacting water molecules. In 4, intermolecular pi center dot center dot center dot pi interactions involving the pyrazolyl rings are relevant. Complexes 2-5 display a high solubility in water (S-25 degrees C ca. 10-12 mg mL(-1)), a favourable feature towards their application as catalysts (or catalyst precursors) for the peroxidative oxidation of cyclo-hexane to cyclohexanol and cyclohexanone, with aqueous H2O2/MeCN, at room temperature (TON values up to ca. 385). (C) 2011 Elsevier B. V. All rights reserved.

Structural characterization and immunogenicity in wildtype and immune tolerant mice of degraded recombinant human interferon alpha2b

Purpose - To study the influence of protein structure on the immunogenicity in wildtype and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). Methods - RhIFNα2b was degraded by metal catalyzed oxidation (M), crosslinking with glutaraldehyde (G), oxidation with hydrogen peroxide (H) and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reversed-phase HPLC, SDS-PAGE, Western blotting and mass spectrometry. The immunogenicity of the products was evaluated in wildtype mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by ELISA or surface plasmon resonance. Results - M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Native (N) rhIFNα2b was immunogenic in the wildtype mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The antirhIFNα2b antibody levels in the wildtype mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ~ N-rhIFNα2b >> B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. Conclusions - RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.

Structural characterization and immunogenicity in wild-type and immune tolerant mice of degraded recombinant human interferon Alpha2b

Purpose: This study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). Methods: RhIFNα2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance. Results: M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNα2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The anti-rhIFNα2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ∼ N-rhIFNα2b ≫ B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. Conclusions: RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.