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We sought to identify novel biomarkers and related mechanisms that might shape the immune infiltration in IDD, thereby providing novel perspective for IDD diagnosis and therapies. Gene expression data sets GSE124272 (for initial analysis) and GSE56081 (for validation analysis) involving samples from IDD patients and healthy controls were retrieved from the Gene Expression Omnibus (GEO) database. Immune genes associated with IDD were identified by GSEA; module genes that exhibited coordinated expression patterns and the strongest positive or negative correlation with IDD were identified by WGCNA. The intersection between immune genes and module genes was used for LASSO variable selection, whereby we obtained pivotal genes that were highly representative of IDD. We then correlated (Pearson correlation) the expression of pivotal genes with immune cell proportion inferred by CIBERSORT algorithm, and revealed the potential immune-regulatory roles of pivotal genes on the pathogenesis of IDD. We discovered several immune-associated pathways in which IDD-associated immune genes were highly clustered, and identified two gene modules that might promote or inhibit the pathogenesis of IDD. These candidate genes were further narrowed down to 8 pivotal genes, namely, MSH2, LY96, ADAM8, HEBP2, ANXA3, RAB24, ZBTB16 and PIK3CD, among which ANXA3, MSH2, ZBTB16, LY96, PIK3CD, ZBTB16, and ADAM8 were revealed to be correlated with the proportion of CD8 T cells and resting memory CD4 T cells. This work identified 8 pivotal genes that might be involved in the pathogenesis of IDD through triggering various immune-associated pathways and altering the composition of immune and myeloid cells in IDD patients, which provides novel perspectives on IDD diagnosis and treatment.

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

Heme (iron protoporphyrin IX) is an essential regulator conserved in all known organisms. We investigated the kinetics of intracellular accumulation of hemin (oxidized form) in human transformed proerythroid K562 cells using [14 C]-hemin and observed that it is time and temperature-dependent, affected by the presence of serum proteins, as well as the amphipathic/hydrophobic properties of hemin. Hemin-uptake exhibited saturation kinetics as a function of the concentration added, suggesting the involvement of a carrier-cell surface receptor-mediated process. The majority of intracellular hemin accumulated in the cytoplasm, while a substantial portion entered the nucleus. Cytosolic proteins isolated by hemin-agarose affinity column chromatography (HACC) were found to form stable complexes with [59 Fe]-hemin. The HACC fractionation and Liquid chromatography-mass spectrometry analysis of cytosolic, mitochondrial, and nuclear protein isolates from K562 cell extracts revealed the presence of a large number of hemin-binding proteins (HeBPs) of diverse ontologies, including heat shock proteins, cytoskeletal proteins, enzymes, and signaling proteins such as actinin a4, mitogen-activated protein kinase 1 as well as several others. The subsequent computational analysis of the identified HeBPs using HemoQuest confirmed the presence of various hemin/heme-binding motifs [C(X)nC, H, Y] in their primary structures and conformations. The possibility that these HeBPs contribute to a heme intracellular trafficking protein network involved in the homeostatic regulation of the pool and overall functions of heme is discussed.

Exposure to lead (Pb), a known toxin causing developmental neurotoxicity, can impair neurogenesis and oxidative phosphorylation (OXPHOS), but the mechanism is not clarified. In the current study, we aim to explore the effects of Pb on the differentiation of SH-SY5Y cells and investigate the role of heme and heme-binding protein BACH1 during differentiation. We found that Pb exposure caused a shift from OXPHOS to glycolysis, resulting in neurogenesis impairment by decreasing neurite growth and downregulation of PSD95 and Synapsin-1 in differentiated SH-SY5Y cells. Heme reduction mediated this mitochondria metabolism repression caused by Pb depending on BACH1 activation. Hemin supplement alleviated Pb-induced OXPHOS damage and adenosine triphosphate (ATP) reduction in differentiated SH-SY5Y cells, and further protected for Pb-induced damage of synapse. Heme binding factor BACH1 was negatively regulated by heme content and BACH1 knockout rescued the Pb-induced transcription and expression decline of genes related to OXPHOS and abrogated Pb-induced growth inhibition of axon promotion and synapse formation. Collectively, the present study demonstrates that heme deficiency mediates OXPHOS damage caused by Pb through BACH1 activation, resulting in neurogenesis impairment.

The NnlA enzyme from Variovorax sp. strain JS1663 degrades the linear nitramine N-nitroglycine (NNG)—a natural product produced by some bacteria—to glyoxylate and nitrite (NO2−). Ammonium (NH4+) was predicted as the third product of this reaction. A source of non-heme FeII was shown to be required for initiation of NnlA activity. However, it was unclear if this FeII was being used as a metallocofactor or a reductant. This study reveals that NnlA contains a b-type heme cofactor. Reduction of this heme is required to initiate NnlA activity. Reduction can occur either by addition of a non-heme FeII source or by reduction with dithionite. Therefore, FeII is not an essential substrate for holoenzyme activity. Data are presented showing that reduced NnlA (FeII-NnlA) can catalyze at least 100 turnovers. In addition, this catalysis occurred in the absence of O2. Finally, NH4+ was verified as the third product, accounting for the complete nitrogen mass balance. Size exclusion chromatography showed that NnlA is a dimer in solution. Additionally, FeII-NnlA is oxidized by O2 and NO2− and binds carbon monoxide (CO) and nitric oxide (NO). These are characteristics shared with PAS domains; NnlA was previously shown to exhibit homology with such domains. Providing further evidence, a structural homology model of NnlA was generated based on the structure of the PAS domain from Pseudomonas aeruginosa Aer2. The structural homology model suggested His73 is the axial ligand of the NnlA heme. Site-directed mutagenesis of His73 to alanine decreased the heme occupancy of NnlA and eliminated NNG activity, providing evidence that the homology model is valid. We conclude that NnlA forms a homodimeric heme-binding PAS domain protein that requires reduction for initiation of the activity.ImportanceLinear nitramines are potential carcinogens. These compounds result from environmental degradation of high-energy cyclic nitramines and as by-products of carbon capture technologies. Mechanistic understanding of the biodegradation of linear nitramines is critical to inform approaches for their remediation. The best understood biodegradation of a linear nitramine is NNG degradation by NnlA from Variovorax sp. strain JS 1663; however, it is unclear why non-heme iron was required to initiate enzymatic turnover. This study shows that non-heme iron is unnecessary. Instead, our study reveals that NnlA contains a heme cofactor, the reduction of which is critical for activating NNG degradation activity. These studies constrain the proposals for NnlA reaction mechanisms, thereby informing mechanistic studies of degradation of anthropogenic nitramine contaminants. In addition, these results will future work to design biocatalysts to degrade these nitramine contaminants.

According to the whole-genome bioinformatics analysis, a heme-binding protein from Nocardia seriolae (HBP) was found. HBP was predicted to be a bacterial secretory protein, located at mitochondrial membrane in eukaryotic cells and have a similar protein structure with the heme-binding protein of Mycobacterium tuberculosis, Rv0203. In this study, HBP was found to be a secretory protein and co-localized with mitochondria in FHM cells. Quantitative analysis of mitochondrial membrane potential value, caspase-3 activity, and transcription level of apoptosis-related genes suggested that overexpression of HBP protein can induce cell apoptosis. In conclusion, HBP was a secretory protein which may target to mitochondria and involve in cell apoptosis in host cells. This research will promote the function study of HBP and deepen the comprehension of the virulence factors and pathogenic mechanisms of N. seriolae.

Purpose Penile erection requires integrative interactions between vascular endothelial cells, pericytes, smooth muscle cells, and autonomic nerves. Furthermore, the importance of the role played by pericytes in the pathogenesis of angiopathy has only recently been appreciated. However, global gene expression in pericytes in diabetes mellitus-induced erectile dysfunction (DMED) remains unclear. We aimed to identify potential target genes related to DMED in mouse cavernous pericytes (MCPs). Materials and Methods Mouse cavernous tissue was allowed to settle under gravity in collagen I-coated dishes, and sprouted cells were subcultivated for experiments. To imitate diabetic conditions, MCPs were treated with normal-glucose (NG, 5 mM) or high-glucose (HG, 30 mM) media for 3 days. Microarray technology was used to evaluate gene expression profiles, and RT-PCR was used to validate sequencing data. Histological examinations and Western blot were used to validate final selected target genes related to DMED. Results Decreased tube formation and increased apoptosis were detected in MCPs exposed to the HG condition. As shown by microarray analysis, the gene expression profiles of MCPs exposed to the NG or HG condition differed. A total of 2,523 genes with significantly altered expression were classified into 15 major gene categories. After further screening based on gene expression and RT-PCR and histologic results, we found that Hebp1 gene expression was significantly diminished under the HG condition and in DM mice. Conclusions This gene profiling study provides new potential targets responsible for diabetes in MCPs. Validation studies suggest that Hebp1 may be a suitable biomarker for DMED., Graphical Abstract

Group A Streptococcus (GAS) is a major pathogen that causes simple and invasive infections. GAS requires iron for metabolic processes and pathogenesis, and heme is its preferred iron source. We previously described the iron-regulated hupZ in GAS, showing that a recombinant HupZ-His6 protein binds and degrades heme. The His6 tag was later implicated in heme iron coordination by HupZ-His6. Hence, we tested several recombinant HupZ proteins, including a tag-free protein, for heme binding and degradation in vitro. We established that HupZ binds heme but without coordinating the heme iron. Heme-HupZ readily accepted exogenous imidazole as its axial heme ligand, prompting degradation. Furthermore, HupZ bound a fragment of heme c (whose iron is coordinated by the cytochrome histidine residue) and exhibited limited degradation. GAS, however, did not grow on a heme c fragment as an iron source. Heterologous HupZ expression in Lactococcus lactis increased heme b iron use. A GAS hupZ mutant showed reduced growth when using hemoglobin as an iron source, increased sensitivity to heme toxicity, and decreased fitness in a murine model for vaginal colonization. Together, the data demonstrate that HupZ contributes to heme metabolism and host survival, likely as a heme chaperone. HupZ is structurally similar to the recently described heme c-degrading enzyme, Pden_1323, suggesting that the GAS HupZ might be divergent to play a new role in heme metabolism.

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2−/− mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin−/− mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs. NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.

Antidepressants are an effective treatment for major depressive disorder (MDD), although individual response is unpredictable and highly variable. Whilst the mode of action of antidepressants is incompletely understood, many medications are associated with changes in DNA methylation in genes that are plausibly linked to their mechanisms. Studies of DNA methylation may therefore reveal the biological processes underpinning the efficacy and side effects of antidepressants. We performed a methylome-wide association study (MWAS) of self-reported antidepressant use accounting for lifestyle factors and MDD in Generation Scotland (GS:SFHS, N = 6428, EPIC array) and the Netherlands Twin Register (NTR, N = 2449, 450 K array) and ran a meta-analysis of antidepressant use across these two cohorts. We found ten CpG sites significantly associated with self-reported antidepressant use in GS:SFHS, with the top CpG located within a gene previously associated with mental health disorders, ATP6V1B2 (β = -0.055, pcorrected = 0.005). Other top loci were annotated to genes including CASP10, TMBIM1, MAPKAPK3, and HEBP2, which have previously been implicated in the innate immune response. Next, using penalised regression, we trained a methylation-based score of self-reported antidepressant use in a subset of 3799 GS:SFHS individuals that predicted antidepressant use in a second subset of GS:SFHS (N = 3360, β = 0.377, p = 3.12 × 10-11, R2 = 2.12%). In an MWAS analysis of prescribed selective serotonin reuptake inhibitors, we showed convergent findings with those based on self-report. In NTR, we did not find any CpGs significantly associated with antidepressant use. The meta-analysis identified the two CpGs of the ten above that were common to the two arrays used as being significantly associated with antidepressant use, although the effect was in the opposite direction for one of them. Antidepressants were associated with epigenetic alterations in loci previously associated with mental health disorders and the innate immune system. These changes predicted self-reported antidepressant use in a subset of GS:SFHS and identified processes that may be relevant to our mechanistic understanding of clinically relevant antidepressant drug actions and side effects., NIHR Senior Investigator Award

Free heme toxicity in the vascular endothelium is critical for the pathogenesis of hemolytic disorders including sickle cell disease. In the current study, it is demonstrated that human alpha1-antitrypsin (A1AT), a serine protease inhibitor with high binding-affinity for heme, rescues endothelial cell (EC) injury caused by free heme. A1AT provided endothelial protection against free heme toxicity via a pathway that differs from human serum albumin and hemopexin, two prototypical heme-binding proteins. A1AT inhibited heme-mediated pro-inflammatory activation and death of ECs, but did not affect the increase in intracellular heme levels and up-regulation of the heme-inducible enzyme heme oxygenase-1. Moreover, A1AT reduced heme-mediated generation of mitochondrial reactive oxygen species. Extracellular free heme led to an increased up-take of A1AT by ECs, which was detected in lysosomes and was found to reduce heme-dependent alkalization of these organelles. Finally, A1AT was able to restore heme-dependent dysfunctional autophagy in ECs. Taken together, our findings show that A1AT rescues ECs from free heme-mediated pro-inflammatory activation, cell death and dysfunctional autophagy. Hence, A1AT therapy may be useful in the treatment of hemolytic disorders such as sickle cell disease., Graphical abstract Image 1

The plateau pika (Ochotona curzoniae) is native to the Qinghai-Tibet Plateau. In this study, the gene that encodes a heme-binding protein in the pulmonary surfactant (PS) of the pika is identified. The protein is a homotetrameric hemoglobin (δ

The Aer2 receptor from Pseudomonas aeruginosa has an O(2)-binding PAS-heme domain that stabilizes O(2) via a Trp residue in the distal heme pocket. Trp rotates ~90° to bond with ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in both the presence and absence of O(2), and the dimer interface was consistent with the isolated PAS dimer structure. O(2)-mediated changes were also consistent with structural predictions where the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O(2)-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS sensing domain.

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

Antidepressants are an effective treatment for major depressive disorder (MDD), although individual response is unpredictable and highly variable. Whilst the mode of action of antidepressants is incompletely understood, many medications are associated with changes in DNA methylation in genes that are plausibly linked to their mechanisms. Studies of DNA methylation may therefore reveal the biological processes underpinning the efficacy and side effects of antidepressants. We performed a methylome-wide association study (MWAS) of self-reported antidepressant use accounting for lifestyle factors and MDD in Generation Scotland (GS:SFHS, N = 6428, EPIC array) and the Netherlands Twin Register (NTR, N = 2449, 450 K array) and ran a meta-analysis of antidepressant use across these two cohorts. We found ten CpG sites significantly associated with self-reported antidepressant use in GS:SFHS, with the top CpG located within a gene previously associated with mental health disorders, ATP6V1B2 (β = -0.055, p

Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation–PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host.

In recent decades, many genome-wide association studies on insomnia have reported numerous genes harboring multiple risk variants. Nevertheless, the molecular functions of these risk variants conveying risk to insomnia are still ill-studied. In the present study, we integrated GWAS summary statistics (N=386,533) with two independent brain expression quantitative trait loci (eQTL) datasets (N=329) to determine whether expression-associated SNPs convey risk to insomnia. Furthermore, we applied numerous bioinformatics analyses to highlight promising genes associated with insomnia risk. By using Sherlock integrative analysis, we detected 449 significant insomnia-associated genes in the discovery stage. These identified genes were significantly overrepresented in six biological pathways including Huntington’s disease (P=5.58 × 10−5), Alzheimer’s disease (P=5.58 × 10−5), Parkinson’s disease (P=6.34 × 10−5), spliceosome (P=1.17 × 10−4), oxidative phosphorylation (P=1.09 × 10−4), and wnt signaling pathways (P=2.07 × 10−4). Further, five of these identified genes were replicated in an independent brain eQTL dataset. Through a PPI network analysis, we found that there existed highly functional interactions among these five identified genes. Three genes of LDHA (P=0.044), DALRD3 (P=5.0 × 10−5), and HEBP2 (P=0.032) showed significantly lower expression level in brain tissues of insomnic patients than that in controls. In addition, the expression levels of these five genes showed prominently dynamic changes across different time points between behavioral states of sleep and sleep deprivation in mice brain cortex. Together, the evidence of the present study strongly suggested that these five identified genes may represent candidate genes and contributed risk to the etiology of insomnia.

Toll-like receptors (TLRs), also known as pattern recognition receptors, respond to exogenous pathogens and to intrinsic danger signals released from damaged cells and tissues. The tetrapyrrole heme has been suggested to be an agonist for TLR4, the receptor for the pro-inflammatory bacterial component lipopolysaccharide (LPS), synonymous with endotoxin. Heme is a double-edged sword with contradictory functions. On the one hand, it has vital cellular functions as the prosthetic group of hemoproteins including hemoglobin, myoglobin, and cytochromes. On the other hand, if released from destabilized hemoproteins, non-protein bound or “free” heme can have pro-oxidant and pro-inflammatory effects, the mechanisms of which are not fully understood. In this review, the complex interactions between heme and TLR4 are discussed with a particular focus on the role of heme-binding serum proteins in handling extracellular heme and its impact on TLR4 signaling. Moreover, the role of heme as a direct and indirect trigger of TLR4 activation and species-specific differences in the regulation of heme-dependent TLR4 signaling are highlighted.

Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIIIhemoprotein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell irond-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.

Made available in DSpace on 2020-12-12T02:39:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The myofibrillar fragmentation index (MFI) is an indicative trait for meat tenderness. Longissimus thoracis muscle samples from the 20 most extreme bulls (out of 80 bulls set) for MFI (high (n = 10) and low (n = 10) groups) trait were used to perform transcriptomic analysis, using RNA Sequencing (RNA-Seq). An average of 24.616 genes was expressed in the Nellore muscle transcriptome analysis. A total of 96 genes were differentially expressed (p value ≤ 0.001) between the two groups of divergent bulls for MFI. The HEBP2 and BDH1 genes were overexpressed in animals with high MFI. The MYBPH and MYL6, myosin encoders, were identified. The differentially expressed genes were related to increase mitochondria efficiency, especially in cells under oxidative stress conditions, and these also were related to zinc and calcium binding, membrane transport, and muscle constituent proteins, such as actin and myosin. Most of those genes were involved in metabolic pathways of oxidation-reduction, transport of lactate in the plasma membrane, and muscle contraction. This is the first study applying MFI phenotypes in transcriptomic studies to identify and understand differentially expressed genes for beef tenderness. These results suggest that differences detected in gene expression between high and low MFI animals are related to reactive mechanisms and structural components of oxidative fibers under the condition of cellular stress. Some genes may be selected as positional candidate genes to beef tenderness, MYL6, MYBPH, TRIM63, TRIM55, TRIOBP, and CHRNG genes. The use of MFI phenotypes could enhance results of meat tenderness studies. School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) Department of Animal Biosciences Centre for Genetic Improvement of Livestock University of Guelph APTA Beef Cattle Center Animal Science Institute National Council for Scientific and Technological Development (CNPq) College of Veterinary and Animal Science São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences São Paulo State University (Unesp) College of Veterinary and Animal Science São Paulo State University (Unesp) FAPESP: # 09/16118-5 FAPESP: #17/04270-3 FAPESP: #18/11154-2

Isoniazid (INH) is a cornerstone of antitubercular therapy. Mycobacterium tuberculosis complex bacteria are the only mycobacteria sensitive to clinically relevant concentrations of INH. All other mycobacteria, including M. marinum and M. avium subsp. paratuberculosis are resistant. INH requires activation by bacterial KatG to inhibit mycobacterial growth. We tested the role of the differences between M. tuberculosis KatG and that of other mycobacteria in INH sensitivity. We cloned the M. bovis katG gene into M. marinum and M. avium subsp. paratuberculosis and measured the MIC of INH. We recombinantly expressed KatG of these mycobacteria and tested in vitro binding to, and activation of, INH. Introduction of katG from M. bovis into M. marinum and M. avium subsp. paratuberculosis rendered them 20 to 30 times more sensitive to INH. Analysis of different katG sequences across the genus found KatG evolution diverged from RNA polymerase-defined mycobacterial evolution. Biophysical and biochemical tests of M. bovis and nontuberculous mycobacteria (NTM) KatG proteins showed lower affinity to INH and substantially lower enzymatic capacity for the conversion of INH into the active form in NTM. The KatG proteins of M. marinum and M. avium subsp. paratuberculosis are substantially less effective in INH activation than that of M. tuberculosis, explaining the relative INH insensitivity of these microbes. These data indicate that the M. tuberculosis complex KatG is divergent from the KatG of NTM, with a reciprocal relationship between resistance to host defenses and INH resistance. Studies of bacteria where KatG is functionally active but does not activate INH may aid in understanding M. tuberculosis INH-resistance mechanisms, and suggest paths to overcome them.

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3′–5′ guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.

International audience; Iron acquisition is an essential aspect of cell physiology for most bacteria. Although much is known about how bacteria initially recognize the various iron sources they can encounter, whether siderophore, heme, host iron/heme binding proteins, much less is known about how the iron containing compounds (Fe 2+ , Fe 3+ , Fe 3+-siderophore complex or heme) are transported across the cytoplasmic membrane. This last transport step is powered by specific ABC (ATP-Binding-Cassette) transporters, made up of a substrate binding protein (SBP) that delivers its cargo to the TMD (TransMembrane Domain) of the ABC transporter triggering the entry of the substrate inside the cytoplasm upon catalytic activity of the ABC module. This review focuses on structural aspects of the functioning of such ABC transporters with the most part devoted to the substrate binding proteins.

A unique π-expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust β-barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N-(1-pyrenyl)maleimide (Pyr) to prepare a series of NB-Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB-Pyr indicates that azachalcone (aza), which is a dienophile for a Diels-Alder (DA) reaction, is efficiently incorporated within the pyrene-linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB-Pyr in the presence of CuII ion in high yield and high enantio- and regioselectivity.

In the HIV-1 replication cycle, the endosomal sorting complex required for transport (ESCRT) machinery promotes viral budding and release in the late stages. In this process, the ESCRT proteins, ALIX and TSG101, are recruited through interactions with HIV-1 Gag p6. ALG-2, also known as PDCD6, interacts with both ALIX and TSG101 and bridges ESCRT-III and ESCRT-I. In this study, we show that ALG-2 affects HIV-1 production negatively at both the exogenous and endogenous levels. Through a yeast two-hybrid screen, we identified HEBP2 as the binding partner of ALG-2, and we solved the crystal structure of the ALG-2·HEBP2 complex. The function of ALG-2·HEBP2 complex in HIV-1 replication was further explored. ALG-2 inhibits HIV-1 production by affecting Gag expression and distribution, and HEBP2 might aid this process by tethering ALG-2 in the cytoplasm.

Pathogenic bacteria remove iron from the haem of host tissues and use it as a catalytic center of many enzymes. Haem uptake by pathogenic bacteria is facilitated by the membrane-integrated haem importer, which belongs to the type II ATP-binding cassette (ABC) transporter. Here we present crystal structures of Burkholderia cenocepacia haem importer BhuUV complexed with the periplasmic haem-binding protein BhuT and in the absence of BhuT. The transmembrane helices of these structures show an inward-facing conformation, in which the cytoplasmic gate of the haem translocation pathway is completely open. Since this conformation is found in both the haem- and nucleotide-free form, the structure of BhuUV-T provides the post-translocation state and the missing piece in the transport cycle of the type II importer. Structural comparison with the outward-facing conformation reported for the haem importer ortholog HmuUV from Yersenia pestis gives mechanistic insights into conformational transitions and haem secretion during the haem transport cycle., Pathogenic bacteria acquire iron from heme cofactors imported by ABC heme transporters. Here the authors present crystal structures of Burkholderia cenocepacia heme importer BhuUV with and without the heme-binding protein BhuT, gathering mechanistic insight into the catalytic cycle of heme import.

Alzheimer’s disease is the most prevalent neurodegenerative disorder leading to progressive cognitive decline. Despite decades of research, understanding AD progression at the molecular level, especially at its early stages, remains elusive. Here, we identified several presymptomatic AD markers by investigating brain proteome changes over the course of neurodegeneration in a transgenic mouse model of AD (3×Tg-AD). We show that one of these markers, heme-binding protein 1 (Hebp1), is elevated in the brains of both 3×Tg-AD mice and patients affected by rapidly-progressing forms of AD. Hebp1, predominantly expressed in neurons, interacts with the mitochondrial contact site complex (MICOS) and exhibits a perimitochondrial localization. Strikingly, wildtype, but not Hebp1-deficient, neurons showed elevated cytotoxicity in response to heme-induced apoptosis. Increased survivability in Hebp1-deficient neurons is conferred by blocking the activation of the mitochondrial-associated caspase signaling pathway. Taken together, our data highlight a role of Hebp1 in progressive neuronal loss during AD progression.

Nutrient iron sequestration is the most significant form of nutritional immunity and causes bacterial pathogens to evolve strategies of host iron scavenging. Cigarette smoking contains iron particulates altering lung and systemic iron homeostasis, which may enhance colonization in the lungs of patients suffering chronic obstructive pulmonary disease (COPD) by opportunistic pathogens such as nontypeable. NTHi is a heme auxotroph, and the NTHi genome contains multiple heme acquisition systems whose role in pulmonary infection requires a global understanding. In this study, we determined the relative contribution to NTHi airway infection of the four heme-acquisition systems HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF that are located at the bacterial outer membrane or the periplasm. Our computational studies provided plausible 3D models for HbpA, SapA, PE, and HxuA interactions with heme. Generation and characterization of single mutants in the hxuCBA, hpe, sapA, and hbpA genes provided evidence for participation in heme binding-storage and inter-bacterial donation. The hxuA, sapA, hbpA, and hpe genes showed differential expression and responded to heme. Moreover, HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF presented moonlighting properties related to resistance to antimicrobial peptides or glutathione import, together likely contributing to the NTHi-host airway interplay, as observed upon cultured airway epithelia and in vivo lung infection. The observed multi-functionality was shown to be system-specific, thus limiting redundancy. Together, we provide evidence for heme uptake systems as bacterial factors that act in a coordinated and multi-functional manner to subvert nutritional- and other sources of host innate immunity during NTHi airway infection., I.R.A. was funded by a PhD studentship from Universidad Pública de Navarra, Spain; S.M. is funded by a postdoctoral contract from CIBERES. This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520- R, from Health Department, Regional Govern from Navarra, Spain, reference 03/2016, and from SEPAR 31/2015 to J.G. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain. This work was also supported by grants from Foundations of Anna and Edwin Berger (KR), the Swedish Medical Research Council (KR: grant number K2015-57X-03163-43-4, www.vr.se), the Cancer Foundation at the University Hospital in Malmö (KR), the Royal Physiographical Society (Forssman’s Foundation) (TAJ), Skåne County Council’s research and development foundation (KR), the Heart Lung Foundation (KR: grant number 20150697, www.hjart-lungfonden.se). The FP7 WeNMR (project# 261572), H2020 West-Life (project# 675858) and the EOSC-hub (project# 777536) European e-Infrastructure projects are acknowledged for the use of their web portals, which make use of the EGI infrastructure with the dedicated support of CESNET-MetaCloud, INFN-PADOVA, NCGINGRID-PT, TW-NCHC, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, Taiwan and the US Open Science Grid

Deviant levels of available heme and related molecules can result from pathological situations such as impaired heme biosynthesis or increased hemolysis as a consequence of vascular trauma or bacterial infections. Heme-related biological processes are affected by these situations, and it is essential to fully understand the underlying mechanisms. While heme has long been known as an important prosthetic group of various proteins, its function as a regulatory and signaling molecule is poorly understood. Diseases such as porphyria are caused by impaired heme metabolism, and heme itself might be used as a drug in order to downregulate its own biosynthesis. In addition, heme-driven side effects and symptoms emerging from heme-related pathological conditions are not fully comprehended and thus impede adequate medical treatment. Several heme-regulated proteins have been identified in the past decades, however, the molecular basis of transient heme-protein interactions remains to be explored. Herein, we summarize the results of an in-depth analysis of heme binding to proteins, which revealed specific binding modes and affinities depending on the amino acid sequence. Evaluating the binding behavior of a plethora of heme-peptide complexes resulted in the implementation of a prediction tool (SeqD-HBM) for heme-binding motifs, which eventually led and will perspectively lead to the identification and verification of so far unknown heme-regulated proteins. This systematic approach resulted in a broader picture of the alternative functions of heme as a regulator of proteins. However, knowledge on heme regulation of proteins is still a bottomless barrel that leaves much scope for future research and development.