Insights into the molecular basis and mechanism of heme-triggered TLR4 signalling: The role of heme-binding motifs in TLR4 and MD2.

Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.

Insights into caudate amphibian skin secretions with a focus on the chemistry and bioactivity of derived peptides.

Amphibians are well-known for their ability to produce and secrete a mixture of bioactive substances in specialized skin glands for the purpose of antibiotic self-protection and defense against predators. Some of these secretions contain various small molecules, such as the highly toxic batrachotoxin, tetrodotoxin, and samandarine. For some time, the presence of peptides in amphibian skin secretions has attracted researchers, consisting of a diverse collection of - to the current state of knowledge - three to 104 amino acid long sequences. From these more than 2000 peptides many are known to exert antimicrobial effects. In addition, there are some reports on amphibian skin peptides that can promote wound healing, regulate immunoreactions, and may serve as antiparasitic and antioxidative substances. So far, the focus has mainly been on skin peptides from frogs and toads (Anura), eclipsing the research on skin peptides of the ca. 700 salamanders and newts (Caudata). Just recently, several novel observations dealing with caudate peptides and their structure-function relationships were reported. This review focuses on the chemistry and bioactivity of caudate amphibian skin peptides and their potential as novel agents for clinical applications.

Heme Determination and Quantification Methods and Their Suitability for Practical Applications and Everyday Use.

Many research institutions, clinical diagnostic laboratories, and blood banks are desperately searching for a possibility to identify and quantify heme in different physiological and pathological settings as well as various research applications. The reasons for this are the toxicity of the heme and the fact that it acts as a hemolytic and pro-inflammatory molecule. Heme only exerts these severe and undesired effects when it is not incorporated in hemoproteins. Upon release from the hemoproteins, it enters a biologically available state (labile heme), in which it is loosely associated with proteins, lipids, nucleic acids, or other molecules. While the current methods and procedures for quantitative determination of heme have been used for many years in different settings, their value is limited by the challenging chemical properties of heme. A major cause of inadequate quantification is the separation of labile and permanently bound heme and its high aggregation potential. Thus, none of the current methods are utilized as a generally applicable, standardized approach. The aim of this Feature is to describe and summarize the most common and frequently used chemical, analytical, and biochemical methods for the quantitative determination of heme. Based on this overview, the most promising approaches for future solutions to heme quantification are highlighted.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme.

BACKGROUND AND AIMS: Blood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. METHODS: We herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. RESULTS: FVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. CONCLUSION: Our study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis.

BACKGROUND: Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. RESULTS: Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 µM) and hemoglobin (∼0.002-17 µM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SIGNIFICANCE: Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.

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.

Oxidation of hemoglobin in the lung parenchyma facilitates the differentiation of pneumococci into encapsulated bacteria.

Pneumococcal pneumonia causes cytotoxicity in the lung parenchyma but the underlying mechanism involves multiple factors contributing to cell death. Here, we discovered that hydrogen peroxide produced by Streptococcus pneumoniae (Spn-H 2 O 2 ) plays a pivotal role by oxidizing hemoglobin, leading to its polymerization and subsequent release of labile heme. At physiologically relevant levels, heme selected a population of encapsulated pneumococci. In the absence of capsule and Spn-H 2 O 2 , host intracellular heme exhibited toxicity towards pneumococci, thus acting as an antibacterial mechanism. Further investigation revealed that heme-mediated toxicity required the ABC transporter GlnPQ. In vivo experiments demonstrated that pneumococci release H 2 O 2 to cause cytotoxicity in bronchi and alveoli through the non-proteolytic degradation of intracellular proteins such as actin, tubulin and GAPDH. Overall, our findings uncover a mechanism of lung toxicity mediated by oxidative stress that favor the growth of encapsulated pneumococci suggesting a therapeutic potential by targeting oxidative reactions. Highlights: Oxidation of hemoglobin by Streptococcus pneumoniae facilitates differentiation to encapsulated pneumococci in vivo Differentiated S. pneumoniae produces capsule and hydrogen peroxide (Spn-H 2 O 2 ) as defense mechanism against host heme-mediated toxicity. Spn-H 2 O 2 -induced lung toxicity causes the oxidation and non-proteolytic degradation of intracellular proteins tubulin, actin, and GAPDH. The ABC transporter GlnPQ is a heme-binding complex that makes Spn susceptible to heme toxicity.

Host and viral proteins involved in SARS-CoV-2 infection differentially bind heme.

In most severe cases, SARS-CoV-2-induced autoimmune reactions have been associated with hemolytic complications. Hemolysis-derived heme from ruptured red blood cells has been shown to trigger a variety of fatal proinflammatory and procoagulant effects, which might deteriorate the progression of COVID-19. In addition, the virus itself can induce proinflammatory signals via the accessory protein 7a. Direct heme binding to the SARS-CoV-2 protein 7a ectodomain and other COVID-19-related proteins has been suggested earlier. Here, we report the experimental analysis of heme binding to the viral proteins spike glycoprotein, protein 7a as well as the host protein ACE2. Thus, protein 7a chemical synthesis was established, including an in-depth analysis of the three different disulfide-bonded isomers. Surface plasmon resonance spectroscopy and in silico studies confirm a transient, biphasic binding behavior, and heme-binding affinities in the nano- to low micromolar range. These results confirm the presence of the earlier identified heme-binding motifs and emphasize the relevance for consideration of labile heme in preexisting or SARS-CoV-2-induced hemolytic conditions in COVID-19 patients.

Exploring the Complex Network of Heme-Triggered Effects on the Blood Coagulation System.

Excess labile heme, occurring under hemolytic conditions, displays a versatile modulator in the blood coagulation system. As such, heme provokes prothrombotic states, either by binding to plasma proteins or through interaction with participating cell types. However, despite several independent reports on these effects, apparently contradictory observations and significant knowledge gaps characterize this relationship, which hampers a complete understanding of heme-driven coagulopathies and the development of suitable and specific treatment options. Thus, the computational exploration of the complex network of heme-triggered effects in the blood coagulation system is presented herein. Combining hemostasis- and heme-specific terminology, the knowledge available thus far was curated and modeled in a mechanistic interactome. Further, these data were incorporated in the earlier established heme knowledge graph, "HemeKG", to better comprehend the knowledge surrounding heme biology. Finally, a pathway enrichment analysis of these data provided deep insights into so far unknown links and novel experimental targets within the blood coagulation cascade and platelet activation pathways for further investigation of the prothrombotic nature of heme. In summary, this study allows, for the first time, a detailed network analysis of the effects of heme in the blood coagulation system.