Heme-binding Protein Research Articles

Association between genetically plasma proteins and osteonecrosis: a proteome-wide Mendelian randomization analysis.

Previous studies have explored the role of plasma proteins on osteonecrosis. This Mendelian randomization (MR) study further assessed plasma proteins on osteonecrosis whether a causal relationship exists and provides some evidence of causality. Summary-level data of 4,907 circulating protein levels were extracted from a large-scale protein quantitative trait loci study including 35,559 individuals by the deCODE Genetics Consortium. The outcome data for osteonecrosis were sourced from the FinnGen study, comprising 1,543 cases and 391,037 controls. MR analysis was conducted to estimate the associations between protein and osteonecrosis risk. Additionally, Phenome-wide MR analysis, and candidate drug prediction were employed to identify potential causal circulating proteins and novel drug targets. We totally assessed the effect of 1,676 plasma proteins on osteonecrosis risk, of which 71 plasma proteins had a suggestive association with outcome risk (P < 0.05). Notably, Heme-binding protein 1 (HEBP1) was significant positively associated with osteonecrosis risk with convening evidence (OR, 1.40, 95% CI, 1.19 to 1.65, P = 3.96 × 10-5, P FDR = 0.044). This association was further confirmed in other MR analysis methods and did not detect heterogeneity and pleiotropy (all P > 0.05). To comprehensively explore the health effect of HEBP1, the phenome-wide MR analysis found it was associated with 136 phenotypes excluding osteonecrosis (P < 0.05). However, no significant association was observed after the false discovery rate adjustment. This comprehensive MR study identifies 71 plasma proteins associated with osteonecrosis, with HEBP1, ITIH1, SMOC1, and CREG1 showing potential as biomarkers of osteonecrosis. Nonetheless, further studies are needed to validate this candidate plasma protein.

Solanum lycopersicum heme-binding protein 2 as a potent antimicrobial weapon against plant pathogens

The rise in antibiotic-resistant bacteria caused by the excessive use of antibiotics has led to the urgent exploration of alternative antimicrobial solutions. Among these alternatives, antimicrobial proteins, and peptides (Apps) have garnered attention due to their wide-ranging antimicrobial effects. This study focuses on evaluating the antimicrobial properties of Solanum lycopersicum heme-binding protein 2 (SlHBP2), an apoplastic protein extracted from tomato plants treated with 1-Methyl tryptophan (1-MT), against Pseudomonas syringae pv. tomato DC3000 (Pst). Computational studies indicate that SlHBP2 is annotated as a SOUL heme-binding family protein. Remarkably, recombinant SlHBP2 demonstrated significant efficacy in inhibiting the growth of Pst within a concentration range of 3–25 μg/mL. Moreover, SlHBP2 exhibited potent antimicrobial effects against other microorganisms, including Xanthomonas vesicatoria (Xv), Clavibacter michiganensis subsp. michiganensis (Cmm), and Botrytis cinerea. To understand the mechanism of action employed by SlHBP2 against Pst, various techniques such as microscopy and fluorescence assays were employed. The results revealed that SlHBP2 disrupts the bacterial cell wall and causes leakage of intracellular contents. To summarize, the findings suggest that SlHBP2 has significant antimicrobial properties, making it a potential antimicrobial agent against a wide range of pathogens. Although further studies are warranted to explore the full potential of SlHBP2 and its suitability in various applications.

Abstract 377: Developing a Novel Oxygen Carrier for Resuscitation and Oxygen Delivery Through Targeted Mutations of Recombinant Heme-Proteins

Introduction: Hemorrhage is a leading cause of death in trauma patients. Interventions dedicated to hemostatic resuscitation have demonstrated benefit in decreasing mortality due to hemorrhagic injury. There remain significant limitations to donor-based blood transfusions. We have identified a heme-binding protein, the Bacterial Regulator of carbon monoxide metabolism (RcoM), with suitable properties to be a robust artificial oxygen carrier. We have engineered a variant that lacks a DNA binding domain and contains cysteine mutations for improved stability (RcoM HBD-CCC). Hypothesis: We hypothesize that engineered RcoM HBD-CCC can be used as an ideal artificial oxygen carrier due to the optimization of 4 factors: ligand binding, nitrite reduction rate, autoxidation rate, and thermal denaturation. Methods: Spectrophotometric methods were used to characterize RcoM HBD-CCC in terms of its biochemical properties for optimal oxygen transport and release. Specifically, ligand binding was tested by determination of the P50 (oxygen pressure for 50% saturation of the molecule) value indicating the affinity of RcoM HBD-CCC towards oxygen. Nitrite reduction assays were carried out to test the rate at which nitric oxide (NO) forms to counteract potential NO scavenging. The autoxidation rate determines how long a protein stays in the reduced state, where it is able to bind to oxygen. Thermal denaturation (T m ) identifies the resistance of a protein to higher temperatures. Results: We determined that RcoM HBD-CCC’s P50 value was 4.2 mmHg which is within the ideal range for oxygen binding affinity given that optimal values for oxygen carriers to mimic hemoglobin oxygen dissociation are between 1 to 26 mmHg. Nitrite reductase rate was 5.6M -1 s -1 which is well above the rate for Hb (K = 0.12 M -1 s -1 ). RcoM HBD-CCC has a low autoxidation rate at 0.016 min -1 . RcoM HBD-CCC has a T m of 71°C which is well above all ambient environmental temperatures. Conclusion: We have designed and synthesized a variant of RcoM that we have demonstrated in in vitro spectrophotometric assays to have ideal biochemical properties for an artificial oxygen carrier. We next plan to test the efficacy of RcoM HBD-CCC in animal models of hemorrhage and assess safety in animal exposure models.

Coagulation Factor XII ( F12) Haploinsufficiency Is Protective Against Venous Thromboembolism in 415,921 Individuals

Background: Coagulation factor XII (FXII) has drawn significant attention as a potential drug target due to the observation that severe congenital deficiency in FXII is not associated with bleeding, while deletion or inhibition of FXII in pre-clinical models consistently protects against thrombosis. However, the contribution of FXII to thrombosis in humans remains controversial, with early reports suggesting that FXII deficiency may even be associated with an increased risk of venous thromboembolism (VTE). Meanwhile, epidemiologic studies of FXII deficiency have yielded conflicting results and are fraught with methodological challenges in part because FXII-deficient patients have no known phenotype and do not typically present to medical attention. To overcome these limitations, we performed the first large-scale analysis of differential thrombotic risk associated with germline loss of function in the gene encoding FXII ( F12) across over 400,000 individuals. Aims: To determine the impact of rare F12 loss-of-function variants on the risk of VTE in a large-scale whole exome sequencing dataset. Methods: The UK Biobank (UKBB) is a population-scale registry dataset containing paired clinical and genomics data for nearly 500,000 individuals. We identified individuals in the UKBB with rare (MAF &amp;lt;1%) variants in F12 that were predicted in silico to result in altered protein function. Qualifying variants included missense variants with an in silico functional impact score of ≥0.9 and high-confidence loss-of-function (HCLOF) variants such as frameshift, nonsense, and essential splice site mutations. We used Firth's logistic regression to assess the association between qualifying F12 variants in a single-gene collapsing analysis and the risk of VTE after adjusting for age, sex, and ancestry as well as several known contributory factors. Differences in the levels of 1,472 plasma proteins between carriers and non-carriers of qualifying F12 variants were evaluated in a subset of 48,892 UKBB participants using the Olink Explore 1536 proteomics platform. Results: Data from 415,921 UKBB participants were included in our analysis. In total, 1,598 individuals carried qualifying variants in F12, of which 99.8% were heterozygous. The presence of rare, function-altering variants in F12 was significantly associated with protection against VTE (OR=0.74, 95% CI: 0.55-0.97, P=0.029) ( Figure 1). This finding remained unchanged when we restricted the analysis to HCLOF variants (OR=0.74, 95% CI: 0.55-0.97, P=0.031). Further, we replicated several known risk factors for VTE, including female sex (P=0.0015), older age (P&amp;lt;1 x 10 -16), higher BMI (P&amp;lt;1 x 10 -16), non-O blood group (P&amp;lt;1 x 10 -16), higher CRP (P&amp;lt;1 x 10 -16), and a history of smoking (P&amp;lt;1 x 10 -16), suggesting that clinical annotation for the dataset is reliable. As expected, the presence of F12 variants was not associated with increased risk of hemorrhage (OR=0.90, P=0.44). Because FXII is also involved in pathways of innate immunity, we interrogated the relationship between carriage of rare F12 variants and sepsis and found no increased risk (OR=0.95, P=0.73). Next, we compared the circulating plasma levels of 1,472 proteins between participants with and without qualifying F12 variants ( Figure 2). We determined that plasma concentrations of three proteins are significantly elevated in participants with a variant F12 allele. Of these, heme binding protein 1 (HEBP1) levels demonstrated the most striking difference (fold-change=7.08, P=1.439 x 10 -43). However, germline loss-of-function variants in the HEBP1 gene are not associated with decreased risk of VTE (OR=1.109, P=0.88), suggesting that HEPB1 levels are regulated by FXII and do not directly influence VTE risk. Conclusions: Operating at the level of germline genetics, our data are the first to demonstrate that FXII loss-of-function is protective against VTE in humans. Because F12 variant carriers in the UKBB dataset predominantly possess only a single defective allele, it is possible that larger effect sizes would be observed among homozygous or compound heterozygous individuals. Additionally, F12 loss-of-function does not appear to increase the risk of hemorrhage or severe infection. Our findings suggest that therapeutic blockade of FXII in humans is likely to be safe and effective.

Double-edged functions of hemopexin in hematological related diseases: from basic mechanisms to clinical application.

It is now understood that hemolysis and the subsequent release of heme into circulation play a critical role in driving the progression of various diseases. Hemopexin (HPX), a heme-binding protein with the highest affinity for heme in plasma, serves as an effective antagonist against heme toxicity resulting from severe acute or chronic hemolysis. In the present study, changes in HPX concentration were characterized at different stages of hemolytic diseases, underscoring its potential as a biomarker for assessing disease progression and prognosis. In many heme overload-driven conditions, such as sickle cell disease, transfusion-induced hemolysis, and sepsis, endogenous HPX levels are often insufficient to provide protection. Consequently, there is growing interest in developing HPX therapeutics to mitigate toxic heme exposure. Strategies include HPX supplementation when endogenous levels are depleted and enhancing HPX's functionality through modifications, offering a potent defense against heme toxicity. It is worth noting that HPX may also exert deleterious effects under certain circumstances. This review aims to provide a comprehensive overview of HPX's roles in the progression and prognosis of hematological diseases. It highlights HPX-based clinical therapies for different hematological disorders, discusses advancements in HPX production and modification technologies, and offers a theoretical basis for the clinical application of HPX.

Enzymatic properties of a non-classical aldoxime dehydratase capable of producing alkyl and arylalkyl nitriles.

Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellaloxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. KEY POINTS: • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate.

Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.

As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR.

Bacterial TANGO2 homologs are heme-trafficking proteins that facilitate biosynthesis of cytochromes c.

Heme, an essential molecule for virtually all living organisms, acts primarily as a cofactor in a large number of proteins. However, how heme is mobilized from the site of synthesis to the locations where hemoproteins are assembled remains largely unknown in cells, especially bacterial ones. In this study, with Shewanella oneidensis as the model, we identified HtpA (SO0126) as a heme-trafficking protein and homolog of TANGO2 proteins found in eukaryotes. We showed that HtpA homologs are widely distributed in all domains of living organisms and have undergone parallel evolution. In its absence, the cytochrome (cyt) c content and catalase activity decreased significantly. We further showed that both HtpA and representative TANGO2 proteins bind heme with 1:1 stoichiometry and a relatively low dissociation constant. Protein interaction analyses substantiated that HtpA directly interacts with the cytochrome c maturation system. Our findings shed light on cross-membrane transport of heme in bacteria and extend the understanding of TANGO2 proteins. IMPORTANCE The intracellular trafficking of heme, an essential cofactor for hemoproteins, remains underexplored even in eukaryotes, let alone bacteria. Here we developed a high-throughput method by which HtpA, a homolog of eukaryotic TANGO2 proteins, was identified to be a heme-binding protein that enhances cytochrome c biosynthesis and catalase activity in Shewanella oneidensis. HtpA interacts with the cytochrome c biosynthesis system directly, supporting that this protein, like TANGO2, functions in intracellular heme trafficking. HtpA homologs are widely distributed, but a large majority of them were found to be non-exchangeable, likely a result of parallel evolution. By substantiating the heme-trafficking nature of HtpA and its eukaryotic homologs, our findings provide general insight into the heme-trafficking process and highlight the functional conservation along evolution in all living organisms.

Shapes and Patterns of Heme-Binding Motifs in Mammalian Heme-Binding Proteins.

Heme is a double-edged sword. On the one hand, it has a pivotal role as a prosthetic group of hemoproteins in many biological processes ranging from oxygen transport and storage to miRNA processing. On the other hand, heme can transiently associate with proteins, thereby regulating biochemical pathways. During hemolysis, excess heme, which is released into the plasma, can bind to proteins and regulate their activity and function. The role of heme in these processes is under-investigated, with one problem being the lack of knowledge concerning recognition mechanisms for the initial association of heme with the target protein and the formation of the resulting complex. A specific heme-binding sequence motif is a prerequisite for such complex formation. Although numerous short signature sequences indicating a particular protein function are known, a comprehensive analysis of the heme-binding motifs (HBMs) which have been identified in proteins, concerning specific patterns and structural peculiarities, is missing. In this report, we focus on the evaluation of known mammalian heme-regulated proteins concerning specific recognition and structural patterns in their HBMs. The Cys-Pro dipeptide motifs are particularly emphasized because of their more frequent occurrence. This analysis presents a comparative insight into the sequence and structural anomalies observed during transient heme binding, and consequently, in the regulation of the relevant protein.

Architectural digest: Thermodynamic stability and domain structure of a consensus monomeric globin

Artificial proteins representing the consensus of a set of homologous sequences have attracted attention for their increased thermodynamic stability and conserved activity. Here, we applied the consensus approach to a b-type heme-binding protein to inspect the contribution of a dissociable cofactor to enhanced stability and the chemical consequences of creating a generic heme environment. We targeted the group 1 truncated hemoglobin (TrHb1) subfamily of proteins for their small size (∼120 residues) and ease of characterization. The primary structure, derived from a curated set of ∼300 representative sequences, yielded a highly soluble consensus globin (cGlbN) enriched in acidic residues. Optical and NMR spectroscopies revealed high-affinity heme binding in the expected site and in two orientations. At neutral pH, proximal and distal iron coordination was achieved with a pair of histidine residues, as observed in some natural TrHb1s, and with labile ligation on the distal side. As opposed to studied TrHb1s, which undergo additional folding upon heme binding, cGlbN displayed the same extent of secondary structure whether the heme was associated with the protein or not. Denaturation required guanidine hydrochloride and showed that apo- and holoprotein unfolded in two transitions—the first (occurring with a midpoint of ∼2 M) was shifted to higher denaturant concentration in the holoprotein (∼3.7 M) and reflected stabilization due to heme binding, while the second transition (∼6.2 M) was common to both forms. Thus, the consensus sequence stabilized the protein but exposed the existence of two separately cooperative subdomains within the globin architecture, masked as one single domain in TrHb1s with typical stabilities. The results suggested ways in which specific chemical or thermodynamic features may be controlled in artificial heme proteins.

Design of Heme Enzymes with a Tunable Substrate Binding Pocket Adjacent to an Open Metal Coordination Site.

The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.

Heme Interactions as Regulators of the Alternative Pathway Complement Responses and Implications for Heme-Associated Pathologies.

Heme (Fe2+-protoporphyrin IX) is a pigment of life, and as a prosthetic group in several hemoproteins, it contributes to diverse critical cellular processes. While its intracellular levels are tightly regulated by networks of heme-binding proteins (HeBPs), labile heme can be hazardous through oxidative processes. In blood plasma, heme is scavenged by hemopexin (HPX), albumin and several other proteins, while it also interacts directly with complement components C1q, C3 and factor I. These direct interactions block the classical pathway (CP) and distort the alternative pathway (AP). Errors or flaws in heme metabolism, causing uncontrolled intracellular oxidative stress, can lead to several severe hematological disorders. Direct interactions of extracellular heme with alternative pathway complement components (APCCs) may be implicated molecularly in diverse conditions at sites of abnormal cell damage and vascular injury. In such disorders, a deregulated AP could be associated with the heme-mediated disruption of the physiological heparan sulphate-CFH coat of stressed cells and the induction of local hemostatic responses. Within this conceptual frame, a computational evaluation of HBMs (heme-binding motifs) aimed to determine how heme interacts with APCCs and whether these interactions are affected by genetic variation within putative HBMs. Combined computational analysis and database mining identified putative HBMs in all of the 16 APCCs examined, with 10 exhibiting disease-associated genetic (SNPs) and/or epigenetic variation (PTMs). Overall, this article indicates that among the pleiotropic roles of heme reviewed, the interactions of heme with APCCs could induce differential AP-mediated hemostasis-driven pathologies in certain individuals.

Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody.

Glaesserella parasuis (G. parasuis.) is the etiological pathogen of Glässer's disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in G. parasuis. In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against recombinant HbpA (rHbpA) of G. parasuis SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of G. parasuis. None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among G. parasuis strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for G. parasuis. Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.

Non-Native Structures of Apomyoglobin and Apoleghemoglobin in Folding Intermediates Related to the Protein Misfolding.

Protein folding is essential for a polypeptide chain to acquire its proper structure and function. Globins are a superfamily of ubiquitous heme-binding α-helical proteins whose function is principally to regulate oxygen homoeostasis. In this review, we explore the hierarchical helical formation in the globin proteins apomyoglobin and leghemoglobin, and we discuss the existence of non-native and misfolded structures occurring during the course of folding to its native state. This review summarizes the research aimed at characterizing and comparing the equilibrium and kinetic intermediates, as well as delineating the complete folding pathway at a molecular level, in order to answer the following questions: "What is the mechanism of misfolding via a folding intermediate? Does the non-native structure stabilize the contemporary intermediate structure? Does the non-native structure induce slower folding?" The role of the non-native structures in the folding intermediate related to misfolding is also discussed.

Ligand-Based Regulation of Dynamics and Reactivity of Hemoproteins.

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme-albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins.

Dupilumab Inhibits Vascular Leakage of Blood Proteins Into Atopic Dermatitis Skin

Atopic dermatitis (AD) skin lesions are associated with oozing, bleeding, and erythema. This suggests AD is associated with vascular changes. Dupilumab is an antibody to the alpha-subunit of interleukin (IL) 4 receptor that demonstrates strong efficacy in the treatment of AD. IL-4 is known to reduce the permeability barrier function of vascular endothelium. To examine the effects of dupilumab on vascular barrier function in AD skin. Using proteomic analysis, we evaluated the plasma protein composition in skin tapes of lesional and non-lesional skin of adults and adolescents with moderate-to-severe AD over the course of a 16-week treatment with dupilumab and compared those to matched healthy subjects. At baseline, 115 plasma proteins were detected in AD skin and globally increased (1.5-fold or greater) compared to healthy skin. Functionally, these proteins included immunoglobulins, proteins involved in the coagulation process, enzymes, protease inhibitors, transport proteins, acute-phase proteins, complement proteins, and other pleiotropic proteins. Noteworthy, fibrinogens, fibronectin, and heme-binding proteins haptoglobin and hemopexin were among the top proteins originating from plasma and were increased in AD lesional versus healthy skin at baseline (p<0.0001). Dupilumab treatment resulted in significantly reduced levels of plasma proteins in AD skin (p<0.0001), with the majority dropping to levels seen in healthy skin or no longer detectable at week 16. Inhibition of IL-4/IL-13 action by dupilumab significantly reduces the efflux of plasma proteins into AD skin. Several of these proteins, such as fibrinogens and fibronectin, are known to enhance Staphylococcus aureuscolonization and are associated with AD skin severity.

Applications of hydrogen deuterium exchange ensemble reweighting to model native protein ensembles, macromolecular interactions and protein drug interactions.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful approach to probe the structural dynamics of a protein in solution. It simultaneously provides time-resolved information on the structural environment of the amide hydrogen of every residue of a protein except Prolines. As such, HDX-MS has been successfully applied to provide insight into the characterization of native state ensembles of proteins, macromolecular interactions, protein-small molecule interactions or protein folding and unfolding studies. However, it remains largely limited to peptide-level resolution information. On the other hand, molecular dynamics (MD) simulations provide a high-resolution view of a protein's structural dynamics by simulating the motion of a protein's atoms over time starting from a high-resolution structure. However, the time scales accessible to MD simulations are limited and can lead to inadequate exploration of a protein's conformational landscape. To bridge this gap, efforts have aimed at developing quantitative and integrative approaches that leverage HDX-MS data to assess and refine computationally generated structural ensembles. HDX ensemble reweighting (HDXer) is one such approach which uses a post hoc maximum entropy approach to adjusts the weights of individual frames of a structural ensemble to conform to HDX-MS data. We had previously applied HDXer to model the native ensemble of the cytoplasmic heme binding protein, PhuS. Here, we have validated the proposed model ensemble by using structure-based drug design approach to successfully target novel, cryptic drug binding sites that would have otherwise not been targetable using HDX-MS or MD simulations alone. Finally, we demonstrate the broader applicability of such HDX-MS guided integrative approach to model macromolecular interactions, missing electron density and protein-small molecule drug interactions

HDX-MS guided ensemble reweighting approach reveals cryptic drug binding sites in the cytoplasmic heme binding protein, PhuS.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a valuable approach to probe protein structure and dynamics. However, interpretation of HDX-MS data is often qualitative and typically limited to peptide-level resolution. Ongoing efforts aim to integrate experimental HDX-MS data with molecular dynamics (MD) approaches, to provide a high-resolution interpretation of the HDX-MS data. One such approach is an HDX-MS based maximum-entropy reweighting approach (HDXer) to reweight computationally generated ensembles to HDX-MS data. Here, HDXer is used in conjunction with enhanced MD (eMD) to characterize the structural dynamics of PhuS. PhuS is a cytoplasmic heme binding protein from P. aeruginosa which shuttles exogenous heme to Heme Oxygenase (HemO) for degradation and is essential for virulence. Through the implementation of a workflow that integrates eMD, HDXer, and dimensional reduction, we had previously modeled the native state ensemble of apo-PhuS revealing a locally diverse apo-PhuS ensemble characterized by large rearrangements of helices surrounding the heme binding. Here, the representative structures from the modeled native ensemble were used as starting points for computer aided drug design (CADD). Implementing the Site Identification by Ligand Competitive Saturation (SILCS) CADD method approximately 1.3 million compounds were screened for each representative structure as well as the published apo PhuS crystal structure, yielding a library of 50 chemically representative candidate compounds. The candidate compounds were then screened using a fluorescence quenching assay. From these assays, several hits were confirmed to bind apo PhuS at nanomolar binding affinities. Most significantly, none of the compounds were derived from CADD using the published apo PhuS crystal structure, demonstrating the utility of HDX-MS guiding ensemble reweighting in revealing cryptic drug binding site in CADD approaches.

Molecular docking analysis of Aza compounds with the heme-binding protein from Tannerella Forsythia.

The high biological activity and interesting optical properties of the aza compounds is known. Therefore, it is of interest to document the molecular docking analysis data of Aza compounds with the heme-binding protein from an anaerobic, Gram-negative bacterium Tannerella Forsythia. Hence, we report the optimal binding features of Aza compounds with the heme-binding protein from Tannerella Forsythia for further consideration in drug discovery against the pathogen.

Molecular docking analysis of oxazole compounds with the heme-binding protein from Porphyromonas gingivalis.

Porphyromonas gingivalis, a peripathogen, has several methods to impede or modify the protective mechanisms of the teeth. Targeting the inhibition of the heme protein will prevent the organism from multiplying and inhibit the virulence mechanism. The literature derived oxazole compounds (1-5) were docked against the protein's active site, and the results show that the selected oxazole derivatives exhibit better interaction compared to clinically proven drugs.