Search Results (26)

Background/Objectives: Biosimilars are central to the modernization of veterinary pharmacology, improving access to complex biological therapies while maintaining quality, safety, and efficacy. Hemoproteins such as cytochrome c, used to support liver function and manage metabolic disorders in animals, are of particular interest. However, their structural complexity and species-specific pharmacology create significant analytical and regulatory challenges for biosimilar development and life-cycle management. Addressing these issues is critical for improving therapeutic outcomes and enabling the broader adoption of biosimilars in veterinary practice. Methods: This narrative review examines the scientific and regulatory principles underlying the development of veterinary biosimilars of hemoproteins, with cytochrome c as a representative model. Regulatory guidelines and relevant scientific literature were analyzed to identify key challenges, knowledge gaps, and required adaptations from human to veterinary medicine, with a focus on biosimilar assessment and life-cycle management. Results: Veterinary biosimilar frameworks are largely informed by EU and US regulatory pathways, emphasizing the stepwise demonstration of biosimilarity through extensive analytical and functional characterization. Long-term safety and efficacy depend on robust Pharmaceutical Quality Systems and effective life-cycle management to ensure manufacturing consistency. For cytochrome c, interchangeability may be acceptable when analytical similarity is exceptionally high. Critical Quality Attributes include polypeptide integrity, heme–protein interaction, iron redox state, and correct three-dimensional conformation. Quality by Design approaches are essential to control manufacturing variability. Despite regional regulatory differences, core scientific principles remain consistent. Conclusions: Hemoprotein biosimilars hold significant promise in veterinary medicine, provided their development is supported by rigorous analytical characterization, strong life-cycle management, and science-based regulatory approaches. Full article

Increasing numbers of reports have revealed novel catalytically active cryptic guanylate cyclases (GCs) and adenylate cyclases (ACs) operating within complex proteins in prokaryotes and eukaryotes. Here we review the structural and functional aspects of some of these cyclases and provide examples that illustrate their roles in the regulation of the intramolecular functions of complex proteins, such as the phytosulfokine receptor (PSKR), and reassess their contribution to signal generation and tuning. Another multidomain protein, Arabidopsis thaliana K⁺ uptake permease (AtKUP5), also harbors multiple catalytically active sites including an N-terminal AC and C-terminal phosphodiesterase (PDE) with an abscisic acid-binding site. We argue that this architecture may enable the fine-tuning and/or sensing of K⁺ flux and integrate hormone responses to cAMP homeostasis. We also discuss how searches with motifs based on conserved amino acids in catalytic centers led to the discovery of GCs and ACs and propose how this approach can be applied to discover hitherto masked active sites in bacterial, fungal, and animal proteomes. Finally, we show that motif searches are a promising approach to discover ancient biological functions such as hormone or gas binding. Full article

Nitric oxide (NO) synthesis, signaling, and scavenging is associated to relevant physiological and pathological events. In all tissues and organs, NO levels and related functions are regulated at different levels, with heme proteins playing pivotal roles. Here, we focus on the structural changes related to the different binding modes of NO to heme-Fe(II), as well as the modulatory effects of this diatomic messenger on heme-protein functions. Specifically, the ability of heme proteins to bind NO at either the distal or proximal side of the heme and the transient interchanging of the binding site is reported. This sheds light on the regulation of O₂ supply to tissues with high metabolic activity, such as the retina, where a precise regulation of blood flow is necessary to meet the demand of nutrients. Full article

The catalytic properties of cytochrome c (Cc) have captured great interest in respect to mitochondrial physiology and apoptosis, and hold potential for novel enzymatic bioremediation systems. Nevertheless, its contribution to the metabolism of environmental toxicants remains unstudied. Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with impactful diseases, and animal models have unveiled concerning signs of PAHs’ toxicity to mitochondria. In this work, a series of eight PAHs with ionization potentials between 7.2 and 8.1 eV were used to challenge the catalytic ability of Cc and to evaluate the effect of vesicles containing cardiolipin mimicking mitochondrial membranes activating the peroxidase activity of Cc. With moderate levels of H₂O₂ and at pH 7.0, Cc catalyzed the oxidation of toxic PAHs, such as benzo[a]pyrene, anthracene, and benzo[a]anthracene, and the cardiolipin-containing membranes clearly increased the PAH conversions. Our results also demonstrate for the first time that Cc and Cc–cardiolipin complexes efficiently transformed the PAH metabolites 2-hydroxynaphthalene and 1-hydroxypyrene. In comparison to horseradish peroxidase, Cc was shown to reach more potent oxidizing states and react with PAHs with ionization potentials up to 7.70 eV, including pyrene and acenaphthene. Spectral assays indicated that anthracene binds to Cc, and docking simulations proposed possible binding sites positioning anthracene for oxidation. The results give support to the participation of Cc in the metabolism of PAHs, especially in mitochondria, and encourage further investigation of the molecular interaction between PAHs and Cc. Full article

The cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synthesis using the peroxide shunt. In this meticulous study, we use hyperfine spectroscopy with a multifrequency approach (X- and Q-band) to characterize in detail the electronic structure of the heme iron of CYP116B5hd in the resting state, which provides structural details about its active site. The hyperfine dipole–dipole interaction between the electron and proton nuclear spins allows for the locating of two different protons from the coordinated water and a beta proton from the cysteine axial ligand of heme iron with respect to the magnetic axes centered on the iron. Additionally, since new anti-cancer therapies target the inhibition of P450s, here we use the CYP116B5hd system—imidazole as a model for studying cytochrome P450 inhibition by an azo compound. The effects of the inhibition of protein by imidazole in the active-site geometry and electron spin distribution are presented. The binding of imidazole to CYP116B5hd results in an imidazole–nitrogen axial coordination and a low-spin heme Fe^III. HYSCORE experiments were used to detect the hyperfine interactions. The combined interpretation of the gyromagnetic tensor and the hyperfine and quadrupole tensors of magnetic nuclei coupled to the iron electron spin allowed us to obtain a precise picture of the active-site geometry, including the orientation of the semi-occupied orbitals and magnetic axes, which coincide with the porphyrin N-Fe-N axes. The electronic structure of the iron does not seem to be affected by imidazole binding. Two different possible coordination geometries of the axial imidazole were observed. The angles between g_x (coinciding with one of the N-Fe-N axes) and the projection of the imidazole plane on the heme were determined to be −60° and −25° for each of the two possibilities via measurement of the hyperfine structure of the axially coordinated ¹⁴N. Full article

Neuronal PAS domain protein 2 (NPAS2) is a hemeprotein comprising a basic helix–loop–helix domain (bHLH) and two heme-binding sites, the PAS-A and PAS-B domains. This protein acts as a pyridine nucleotide-dependent and gas-responsive CO-dependent transcription factor and is encoded by a gene whose expression fluctuates with circadian rhythmicity. NPAS2 is a core cog of the molecular clockwork and plays a regulatory role on metabolic pathways, is important for the function of the central nervous system in mammals, and is involved in carcinogenesis as well as in normal biological functions and processes, such as cardiovascular function and wound healing. We reviewed the scientific literature addressing the various facets of NPAS2 and framing this gene/protein in several and very different research and clinical fields. Full article

Electrochemical biosensors are widely used in a multitude of applications, such as medical, nutrition, research, among other fields. These sensors have been historically used and have not undergone many changes in terms of the involved electrochemical processes. In this work, we propose a new approach on the immobilization and enhancement of the electrochemical properties of the sensing layers through the control and bioconjugation of hemoproteins (hemoglobin, myoglobin, and cytochrome C) on anisotropic gold nanoparticles (gold nanotriangles (AuNTs)). The hemeproteins and the AuNTs are mixed in a solution, resulting in stable bioconjugates that are deposited onto the electrode surface to obtain the biosensors. All the systems proposed herein exhibited direct well-defined redox responses, highlighting the key role of the AuNTs acting as mediators of such electron transfers. Several protein layers surrounding the AuNTs are electroactive, as demonstrated from the charge measured by cyclic voltammetry. The retention of the stability of the hemeproteins once they are part of the bioconjugates is evidenced towards the electrocatalytic reduction of hydrogen peroxide, oxygen, and nitrite. The parameters obtained for the proposed biosensors are similar or even lower than those previously reported for similar systems based on nanomaterials, and they exhibit attractive properties that make them potential candidates for the latest developments in the field of sensing devices. Full article

Hemeprotein detection has motivated extensive research on the direct reaction of a heme molecule and a redox dye. The present study used methylene blue as both donor and acceptor for a redox reaction. First, the solid phases of methylene blue (MB) and graphene (GP) formed a π-π interaction bond at the aromatic rings. The conductivity of GP was better than that of carbon in a carbon electrode (CE). Then, the working CE was modified using strong adsorption of MB/GP on the electrode surface. The surface of the electrode was investigated using a modified and an unmodified electrode. The electrode’s properties were studied using voltammograms of redox couple K₃[Fe(CN)₆]^3−/4−. Its reaction was used to find the active area of the modified electrode, which was 1.76 times bigger than that of the unmodified electrode. The surface coverage values of the modified and unmodified electrodes were 8.17 × 10⁻⁶ and 1.53 × 10⁻⁵ mol/cm², respectively. This research also studied the application of hemeprotein detection. Hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt. C) were studied by the reaction of Fe (III/II) at the heme-redox center. The electrocatalytic reaction between MB/GP and hemeproteins produced an anodic peak at 0.35 V for Hb, Mb, and Cyt. C. This nanohybrid film enhanced electron transfer between protein molecules and the modified carbon electrode. The amperometric measurements show that the limit of detection was 0.2 µM, 0.3 µM, and 0.1 µM for Hb, Mb, and Cyt. C, respectively. The measurement spanned a linear range of 0.2 µM to 5 µM, 0.3 µM to 5 µM, and 0.1 µM to 0.7 µM for Hb, Mb, and Cyt. C, respectively. Hb showed the lowest sensitivity compared with Mb and Cyt. C due to the role of steric hindrance in the hemeprotein specificity structure. This study offers a simple and efficient fabrication platform for electrochemical sensors for hemeproteins. When compared to other complex immobilization processes, the fabrication method for this sensor has many benefits, including no need for special chemicals and easy preparation and electrode modification—both of which are crucial for the development of electrochemical sensing devices. Full article

Heme proteins are known to perform a plethora of biologically important functions. This article reviews work that has been conducted on various class I cytochrome c proteins over a period of nearly 50 years. The article focuses on the relevance of symmetry-lowering heme–protein interactions that affect the function of the electron transfer protein cytochrome c. The article provides an overview of various, mostly spectroscopic studies that explored the electronic structure of the heme group in these proteins and how it is affected by symmetry-lowering deformations. In addition to discussing a large variety of spectroscopic studies, the article provides a theoretical framework that should enable a comprehensive understanding of the physical chemistry that underlies the function not only of cytochrome c but of all heme proteins. Full article

Nitric oxide (NO) is an essential signaling molecule present in most living organisms including bacteria, fungi, plants, and animals. NO participates in a wide range of biological processes including vasomotor tone, neurotransmission, and immune response. However, NO is highly reactive and can give rise to reactive nitrogen and oxygen species that, in turn, can modify a broad range of biomolecules. Much evidence supports the critical role of NO in the virulence and replication of viruses, bacteria, protozoan, metazoan, and fungi, thus representing a general mechanism of host defense. However, pathogens have developed different mechanisms to elude the host NO and to protect themselves against oxidative and nitrosative stress. Here, the strategies evolved by viruses, bacteria, protozoan, metazoan, and fungi to escape from the NO-based host defense are overviewed. Full article

Nitrobindins (Nbs) are all-β-barrel heme proteins and are present in prokaryotes and eukaryotes. Although their function(s) is still obscure, Nbs trap NO and inactivate peroxynitrite. Here, the kinetics of peroxynitrite scavenging by ferric Danio rerio Nb (Dr-Nb(III)) in the absence and presence of CO₂ is reported. The Dr-Nb(III)-catalyzed scavenging of peroxynitrite is facilitated by a low pH, indicating that the heme protein interacts preferentially with peroxynitrous acid, leading to the formation of nitrate (~91%) and nitrite (~9%). The physiological levels of CO₂ dramatically facilitate the spontaneous decay of peroxynitrite, overwhelming the scavenging activity of Dr-Nb(III). The effect of Dr-Nb(III) on the peroxynitrite-induced nitration of L-tyrosine was also investigated. Dr-Nb(III) inhibits the peroxynitrite-mediated nitration of free L-tyrosine, while, in the presence of CO₂, Dr-Nb(III) does not impair nitro-L-tyrosine formation. The comparative analysis of the present results with data reported in the literature indicates that, to act as efficient peroxynitrite scavengers in vivo, i.e., in the presence of physiological levels of CO₂, the ferric heme protein concentration must be higher than 10⁻⁴ M. Thus, only the circulating ferric hemoglobin levels appear to be high enough to efficiently compete with CO₂/HCO₃⁻ in peroxynitrite inactivation. The present results are of the utmost importance for tissues, like the eye retina in fish, where blood circulation is critical for adaptation to diving conditions. Full article

Hydrogen sulfide has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious and neuropathological diseases have been undertaken. Here we review the possible direct targets of H₂S in mammals. H₂S directly interacts with reactive oxygen/nitrogen species and is involved in redox signaling. H₂S also reacts with hemeproteins and modulates metal-containing complexes. Once being oxidized, H₂S can persulfidate proteins by adding -SSH to the amino acid cysteine. These direct modifications by H₂S have significant impact on cell structure and many cellular functions, such as tight junctions, autophagy, apoptosis, vesicle trafficking, cell signaling, epigenetics and inflammasomes. Therefore, we conclude that H₂S is involved in many important cellular and physiological processes. Compounds that donate H₂S to biological systems can be developed as therapeutics for different diseases. Full article

The interaction of heme with blood serum proteins plays an important role in many physiological and pathological processes involving enzyme activity, gene expression and cell proliferation. The mechanisms underlying these interactions are; however, not yet fully understood. New analytical methods able to investigate protein-heme binding in native, biologically representative conditions are thus required. In this work, we present a method based on miniaturized, hollow-fiber flow field-flow fractionation with multiple spectrophotometric and light-scattering detection for size separation of high-abundance serum proteins and selective detection of heme-bound subpopulations. Heme is found to mainly interact with serum albumin, whereas a low amount also binds to other proteins such as IgM. The ability to bind heme in physiological conditions is also investigated for individual serum proteins. IgG is found unable to bind heme at clinically relevant concentrations. The proposed method allows separation, quantitation, and mass/size characterization of serum high-abundance proteins, providing information of heme-protein complex stability and preferred heme-clearing pathways. The same approach could be in perspective extended to the investigation of specific heme-antibody binding, and to further studies involving other molecules of pharmaceutical/clinical interest. Full article

Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive heme enzyme for its significant function in cancer immunotherapy. Potent IDO1 inhibitors have been discovered for decades, whereas no clinical drugs are used for cancer treatment up to now. With the goal of developing medically valuable IDO inhibitors, we performed a systematic study of SAR405838 analogs with a spiro-oxindole skeleton in this study. Based on the expression and purification of human IDO1, the inhibitory activity of spiro-oxindole skeleton compounds to IDO1 was evaluated by IC₅₀ and K_i values. The results demonstrated that inhibitor 3 exhibited the highest IDO1 inhibitory activity with IC₅₀ at 7.9 μM among all inhibitors, which is ~six-fold of the positive control (4−PI). Moreover, inhibitor 3 was found to have the most effective inhibition of IDO1 in MCF-7 cancer cells without toxic effects. Molecular docking analysis revealed that the hydrophobic interaction stabilized the binding of inhibitor 3 to the IDO1 active site and made an explanation for the uncompetitive mode of inhibitors. Therefore, this study provides valuable insights into the screen of more potent IDO1 inhibitors for cancer immunotherapy. Full article

The immune function is closely related to iron (Fe) homeostasis and allostasis. The aim of this bioinformatics-assisted review was twofold; (i) to update the current knowledge of Fe metabolism and its relationship to the immune system, and (ii) to perform a prediction analysis of regulatory network hubs that might serve as potential biomarkers during stress-induced immunosuppression. Several literature and bioinformatics databases/repositories were utilized to review Fe metabolism and complement the molecular description of prioritized proteins. The Search Tool for the Retrieval of Interacting Genes (STRING) was used to build a protein-protein interactions network for subsequent network topology analysis. Importantly, Fe is a sensitive double-edged sword where two extremes of its nutritional status may have harmful effects on innate and adaptive immunity. We identified clearly connected important hubs that belong to two clusters: (i) presentation of peptide antigens to the immune system with the involvement of redox reactions of Fe, heme, and Fe trafficking/transport; and (ii) ubiquitination, endocytosis, and degradation processes of proteins related to Fe metabolism in immune cells (e.g., macrophages). The identified potential biomarkers were in agreement with the current experimental evidence, are included in several immunological/biomarkers databases, and/or are emerging genetic markers for different stressful conditions. Although further validation is warranted, this hybrid method (human-machine collaboration) to extract meaningful biological applications using available data in literature and bioinformatics tools should be highlighted. Full article