Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation.

BACKGROUND: α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme. METHODS: Four A1M-variants were expressed: with cysteine to serine substitution in position 34, lysine to threonine substitutions in positions (92, 118, 130), histidine to serine substitution in position 123 and a wt without mutations. Heme binding was investigated by tryptophan fluorescence quenching, UV-Vis spectrophotometry, circular dichroism, SPR, electrophoretic migration shift, gel filtration, catalase-like activity and molecular simulation. RESULTS: All A1M-variants bound to heme. Mutations in C34, H123 or K(92, 118, 130) resulted in significant absorbance changes, CD spectral changes, and catalase-like activity, suggesting involvement of these side-groups in coordination of the heme-iron. Molecular simulation support a model with two heme-binding sites in A1M involving the mutated residues. Binding of the first heme induces allosteric stabilization of the structure predisposing for a better fit of the second heme. CONCLUSIONS: The results suggest that one heme-binding site is located in the lipocalin pocket and a second binding site between loops 1 and 4. Reactions with the hemes involve the side-groups of C34, K(92, 118, 130) and H123. GENERAL SIGNIFICANCE: The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M.

Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses.

Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza.

Emergency department clinicians' views on implementing psychosocial care following acute paediatric injury: a qualitative study.

Introduction: The early post-trauma period is a key time to provide psychological support to acutely injured children. This is often when they present to emergency departments (EDs) with their families. However, there is limited understanding of the feasibility of implementing psychological support for children and their families in EDs. The aim of this study was to explore UK and Irish ED clinicians' perspectives on developing and implementing psychosocial care which educates families on their children's post-trauma psychological recovery.Methods: Semi-structured individual and group interviews were conducted with 24 UK and Irish ED clinicians recruited via a paediatric emergency research network.Results: Clinicians expressed that there is value in offering psychological support for injured children and their families; however, there are barriers which can prevent this from being effectively implemented. Namely, the prioritisation of physical health, time constraints, understaffing, and a lack of training. Therefore, a potential intervention would need to be brief and accessible, and all staff should be empowered to deliver it to all families.Conclusion: Overall, participants' views are consistent with trauma-informed approaches where a psychosocial intervention should be able to be implemented into the existing ED system and culture. These findings can inform implementation strategies and intervention development to facilitate the development and delivery of an accessible digital intervention for acutely injured children and their families.

The emergency department provides an opportunity for early trauma-informed care for acutely injured children and their families.Addressing psychological distress in emergency care for acutely injured children and their families should adopt a universal trauma-informed approach.The development of a paediatric trauma-informed intervention should consider barriers which can impact implementation into emergency care. Particular barriers highlighted by clinicians include staff shortages, time constraints, and high caseloads.

Functional determinants of human enteric α-defensin HD5: crucial role for hydrophobicity at dimer interface.

Human α-defensins are cationic peptides that self-associate into dimers and higher-order oligomers. They bind protein toxins, such as anthrax lethal factor (LF), and kill bacteria, including Escherichia coli and Staphylococcus aureus, among other functions. There are six members of the human α-defensin family: four human neutrophil peptides, including HNP1, and two enteric human defensins, including HD5. We subjected HD5 to comprehensive alanine scanning mutagenesis. We then assayed LF binding by surface plasmon resonance, LF activity by enzyme kinetic inhibition, and antibacterial activity by the virtual colony count assay. Most mutations could be tolerated, resulting in activity comparable with that of wild type HD5. However, the L29A mutation decimated LF binding and bactericidal activity against Escherichia coli and Staphylococcus aureus. A series of unnatural aliphatic and aromatic substitutions at position 29, including aminobutyric acid (Abu) and norleucine (Nle) correlated hydrophobicity with HD5 function. The crystal structure of L29Abu-HD5 depicted decreased hydrophobic contacts at the dimer interface, whereas the Nle-29-HD5 crystal structure depicted a novel mode of dimerization with parallel ß strands. The effect of mutating Leu(29) is similar to that of a C-terminal hydrophobic residue of HNP1, Trp(26). In addition, in order to further clarify the role of dimerization in HD5 function, an obligate monomer was generated by N-methylation of the Glu(21) residue, decreasing LF binding and antibacterial activity against S. aureus. These results further characterize the dimer interface of the α-defensins, revealing a crucial role of hydrophobicity-mediated dimerization.

Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance.

BACKGROUND: Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states. SCOPE OF RESEARCH: We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout. MAJOR CONCLUSIONS: Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation. GENERAL SIGNIFICANCE: This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies.

Heme binding to human alpha-1 proteinase inhibitor.

BACKGROUND: Heme is a unique prosthetic group of various hemoproteins that perform diverse biological functions; however, in its free form heme is intrinsically toxic in vivo. Due to its potential toxicity, heme binding to plasma proteins is an important safety issue in regard to protein therapeutics derived from human blood. While heme binding by hemopexin, albumin and α(1)-microglobulin has been extensively studied, the role of other plasma proteins remains largely unknown. METHODS: We examined two acute-phase plasma proteins, haptoglobin (Hp) and alpha-1 proteinase inhibitor (α(1)-PI) for possible interactions with heme and bilirubin (BR), the final product of heme degradation, using various techniques: UV/Vis spectroscopy, fluorescence, circular dichroism (CD), and surface plasmon resonance (SPR). RESULTS: According to our data, Hp exhibits a very weak association with both heme and BR; α(1)-PI's affinity to BR is also very low. However, α(1)-PI's affinity to heme (K(D) 2.0×10(-8)M) is of the same order of magnitude as that of albumin (1.26×10(-8)M). The data for α(1)-PI binding with protoporphyrin IX (PPIX) suggest that the elimination of the iron atom from the porphyrin structure results in almost 350-fold lower affinity (K(D) 6.93×10(-6)M), thus indicating that iron is essential for the heme coordination with the α(1)-PI. CONCLUSIONS: This work demonstrates for the first time that human α(1)-PI is a heme binding protein with an affinity to heme comparable to that of albumin. GENERAL SIGNIFICANCE: Our data may have important implications for safety and efficacy of plasma protein therapeutics.

Trp-26 imparts functional versatility to human alpha-defensin HNP1.

We performed a comprehensive alanine scan of human alpha-defensin HNP1 and tested the ability of the resulting analogs to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind human immunodeficiency virus-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild type HNP1 and to undergo further self-association. Thus, the hydrophobicity of residue 26 is not only a key determinant of the direct interactions of HNP1 with target molecules, but it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.

Supporting disengaged children and young people living with diabetes to self-care: a qualitative study in a socially disadvantaged and ethnically diverse urban area.

OBJECTIVE: To explore how to enhance services to support the self-care of children and young people (CYP) clinically considered 'disengaged' by diabetes services. DESIGN: Qualitative study. SETTING: Two diabetes clinics in an ethnically diverse and socially disadvantaged urban area in the UK. Eligible participants were CYP living with type 1 or type 2 diabetes aged between 10 and 25 years who did not attend their last annual hospital appointment. PARTICIPANTS: 22 CYP (14 female and 8 male) aged between 10 and 19 years old took part. The sample was diverse in terms of ethnicity, age at diagnosis, family composition and presence of diabetes among other family members. DATA COLLECTION: Semistructured interviews. DATA ANALYSIS: Data were analysed thematically. RESULTS: Analysis of participant accounts confirmed the crucial importance of non-medicalised care in CYP diabetes care. A life plan was considered as important to participants as a health plan. Participants valued the holistic support provided by friends, family members and school teachers. However, they found structural barriers in their health and educational pathways as well as disparities in the quality of support at critical moments along the life course. They actively tried to maximise their well-being by balancing life priorities against diabetes priorities. Combined, these features could undermine participants engagement with health services where personal strategies were often held back or edited out of clinical appointments in fear of condemnation. CONCLUSION: We demonstrate why diabetes health teams need to appreciate the conflicting pressures experienced by CYP and to coproduce more nuanced health plans for addressing their concerns regarding identity and risk taking behaviours in the context of their life-worlds. Exploring these issues and identifying ways to better support CYP to address them more proactively should reduce disengagement and set realistic health outcomes that make best use of medical resources.

Mental health and wellbeing implications of the COVID-19 quarantine for disabled and disadvantaged children and young people: evidence from a cross-cultural study in Zambia and Sierra Leone.

BACKGROUND: The mental health impact of the COVID-19 pandemic and quarantining on children and young people (CYP) living in low- and middle-income countries (LMICs) has yet to be fully comprehended. CYP in LMICs are at utmost risk, given the COVID-19-related restrictions and social distancing measures, resulting in reduced access to school-based services for nutritional and mental health needs. This study examined mental health of CYP during the first COVID-19 lockdown in Zambia and Sierra Leone. METHOD: A total of 468 disabled and disadvantaged CYP aged 12 to 25 completed a planning tool that comprised the short Warwick-Edinburgh Mental Wellbeing Scale (SWEMWBS), as well as open-ended questions covering social connectedness, physical distancing and educational challenges during the lockdown. The community coaches screened individuals and families who could be eligible to receive emergency aid, and based on a convenience sample following distribution of aid, recipients were invited to complete the planning tool. RESULTS: The data showed that participants in the global south have increasing anxieties and fears centred on accessing offline educational resources and income loss in the family effecting food security and their ability to return to education. Mean (SD) SWEMWBS scores for all participants in Zambia and Sierra Leone, were 19.61 (3.45) and 21.65 (2.84), respectively. Mental well-being scores were lower in females, children aged 12-14 and participants with two or more disabilities. Factors significantly associated with poor mental wellbeing in the sample were: type of disability, nationality, peer relationships, connection to others during the pandemic, knowledge about COVID-19, worry about the long-term impact of COVID-19, and the types of self-isolating. CONCLUSION: The study shows that participants who self-reported low levels of COVID-19 health literacy also scored low on the mental wellbeing self-assessment. Yet, despite undoubted limited resources, these CYP are doing well in identifying their needs and maintaining hope in the face of the problems associated with COVID-19 in countries where stigma persists around mental ill-health.

Characterization of heme binding to recombinant α1-microglobulin.

BACKGROUND: Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions. METHODS: To examine heme binding properties of A1M we utilized UV/Vis absorption spectroscopy, visible circular dichroism (CD), catalase-like activity, migration shift electrophoresis, and surface plasmon resonance (SPR), which was specifically developed for the assessment of His-tagged A1M. RESULTS: The results of this study confirm that A1M is a heme binding protein that can accommodate heme at more than one binding site and/or in coordination with different amino acid residues depending upon heme concentration and ligand-to-protein molar ratio. UV/Vis titration of A1M with heme revealed an unusually large bathochromic shift, up to 38 nm, observed for heme binding to a primary binding site. UV/Vis spectroscopy, visible CD and catalase-like activity suggested that heme is accommodated inside His-tagged (tgA1M) and tagless A1M (ntA1M) in a rather similar fashion although the His-tag is very likely involved into coordination with iron of the heme molecule. SPR data indicated kinetic rate constants and equilibrium binding constants with KD values in a µM range. CONCLUSIONS: This study provided experimental evidence of the A1M heme binding properties by aid of different techniques and suggested an analytical methodology for a rapid evaluation of ligand-binding properties of recombinant A1M versions, also suitable for other His-tagged proteins.

Oversulfated chondroitin sulfate binds to chemokines and inhibits stromal cell-derived factor-1 mediated signaling in activated T cells.

Oversulfated chondroitin sulfate (OSCS), a member of the glycosaminoglycan (GAG) family, was a contaminant in heparin that was linked to the 2008 heparin adverse events in the US. Because of its highly negative charge, OSCS can interact with many components of the contact and immune systems. We have previously demonstrated that OSCS inhibited the complement classical pathway by binding C1 inhibitor and potentiating its interaction with C1s. In the present study, by using surface plasmon resonance, we found OSCS interacts with T cell chemokines that can impact adaptive immunity. The binding of OSCS to stromal cell-derived factor-1 (SDF-1) chemokines, SDF-1α and SDF-1ß, caused a significant change in the secondary structures of these chemokines as detected by far-ultraviolet circular dichroism spectra analysis. Functionally, OSCS binding profoundly inhibited SDF-1-induced calcium mobilization and T cell chemotaxis. Imaging flow cytometry revealed T cell morphological changes mediated by SDF-1α were completely blocked by OSCS. We conclude that the OSCS, a past contaminant in heparin, has broad interactions with the components of the human immune system beyond the contact and complement systems, and that may explain, in part, prior OSCS-related adverse events, while suggesting potentially useful therapeutic applications for related GAGs in the control of inflammation.

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine.

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the ß-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 µg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 µg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 µg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 µg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

Oversulfated chondroitin sulfate inhibits the complement classical pathway by potentiating C1 inhibitor.

Oversulfated chondroitin sulfate (OSCS) has become the subject of multidisciplinary investigation as a non-traditional contaminant in the heparin therapeutic preparations that were linked to severe adverse events. In this study, it was found that OSCS inhibited complement fixation on bacteria and bacterial lysis mediated by the complement classical pathway. The inhibition of complement by OSCS is not due to interference with antibody/antigen interaction or due to consumption of C3 associated with FXII-dependent contact system activation. However, OSCS complement inhibition is dependent on C1 inhibitor (C1inh) since the depletion of C1inh from either normal or FXII-deficient complement plasma prevents OSCS inhibition of complement activity. Surface plasmon resonance measurements revealed that immobilized C1inhibitor bound greater than 5-fold more C1s in the presence of OSCS than in presence of heparin. Although heparin can also inhibit complement, OSCS and OSCS contaminated heparin are more potent inhibitors of complement. Furthermore, polysulfated glycosaminoglycan (PSGAG), an anti-inflammatory veterinary medicine with a similar structure to OSCS, also inhibited complement in the plasma of dogs and farm animals. This study provides a new insight that in addition to the FXII-dependent activation of contact system, oversulfated and polysulfated chondroitin-sulfate can inhibit complement activity by potentiating the classical complement pathway regulator C1inh. This effect on C1inh may play a role in inhibiting inflammation as well as impacting bacterial clearance.

Selective arginines are important for the antibacterial activity and host cell interaction of human alpha-defensin 5.

Defensins constitute a major family of natural antimicrobial peptides that protect the host against microbial invasion. Here, we report on the antibacterial properties and cellular interaction of Human Defensin 5 as a function of its positive charge and hydrophobicity. We find that selective replacement of arginine residues in HD-5 by alanine or charge-neutral lysine residues reduces antibacterial killing as well as host cell interaction. We identify arginines at positions 9 and 28 in the HD-5 sequence as particularly important for its function. Replacement of arginine at position 13 to Histidine, as observed in a Crohn's disease patient, reduced bacterial killing strain-selectively. Finally, we find that HD-5 interacts with host cells via receptor-mediated mechanisms.

The conserved salt bridge in human alpha-defensin 5 is required for its precursor processing and proteolytic stability.

Mammalian alpha-defensins, expressed primarily in leukocytes and epithelia, play important roles in innate and adaptive immune responses to microbial infection. Six invariant cysteine residues forming three indispensable disulfide bonds and one Gly residue required structurally for an atypical beta-bulge are totally conserved in the otherwise diverse sequences of all known mammalian alpha-defensins. In addition, a pair of oppositely charged residues (Arg/Glu), forming a salt bridge across a protruding loop in the molecule, is highly conserved. To investigate the structural and functional roles of the conserved Arg(6)-Glu(14) salt bridge in human alpha-defensin 5 (HD5), we chemically prepared HD5 and its precursor proHD5 as well as their corresponding salt bridge-destabilizing analogs E14Q-HD5 and E57Q-proHD5. The Glu-to-Gln mutation, whereas significantly reducing the oxidative folding efficiency of HD5, had no effect on the folding of proHD5. Bovine trypsin productively and correctly processed proHD5 in vitro but spontaneously degraded E57Q-proHD5. Significantly, HD5 was resistant to trypsin treatment, whereas E14Q-HD5 was highly susceptible. Further, degradation of E14Q-HD5 by trypsin was initiated by the cleavage of the Arg(13)-Gln(14) peptide bond in the loop region, a catastrophic proteolytic event resulting directly in quick digestion of the whole defensin molecule. The E14Q mutation did not alter the bactericidal activity of HD5 against Staphylococcus aureus but substantially enhanced the killing of Escherichia coli. By contrast, proHD5 and E57Q-proHD5 were largely inactive against both strains at the concentrations tested. Our results confirm that the primary function of the conserved salt bridge in HD5 is to ensure correct processing of proHD5 and subsequent stabilization of mature alpha-defensin in vivo.