ABSTRACT. All oxygen binding heme proteins possess enzymic activities. Some of these are reported to have physiological roles (e.g. NO dioxygenases, nitrite reductases), while others are pathophysiological in their consequences. We discuss here the most important of these, which is the peroxidatic activity initiated by the reaction of the heme group with hydrogen peroxide that yields a highly oxidizing oxy-ferryl species and a free radical. Both of these, individually or in combination, can oxidize biological molecules leading to cellular oxidative stress. The evidence that such reactions take place in vivo comes from detection of a number of biomarkers and will be reviewed. These reactions can take place within the cellular environment but because the cellular anti-oxidative defence systems are efficient the extent to which they occur is limited. Once, however, myoglobin or hemoglobin escape their cellular environments and are away from these defences the picture is very different. Following crush injuries that release myoglobin into the blood stream and consequently the kidney (rhabdomyolysis) or sub-arachnoid haemorrhage that allows hemoglobin entry to the cerebral spinal fluid, oxidation of lipids take place to form potent vasoconstrictors e.g. isoprostanes or neuroprostanes. In the course of catalysing these reactions some of the heme proteins themselves are modified forming a covalent bond between the heme group and the protein. The presence of these species provide biomarkers that are unequivocal evidence for the peroxidatic activity. How the pathological consequences flow from these reactions and how therapeutic interventions can be devised to defend against them will be reviewed. The lessons learnt from these studies have informed the design of the next generation of hemoglobin based “blood substitutes” and will also be discussed.

ABSTRACT. How do novel protein functions evolve in an incremental, step-by-step fashion, and to what extent is the gain of new function mechanistically coupled with the loss of ancestral function? These are fundamental questions in molecular evolution and they can be addressed with a protein-engineering approach that permits the identification and functional characterization of causative mutations. Here we report discoveries regarding the molecular basis of a key physiological innovation during vertebrate evolution. We used ancestral protein resurrection in conjunction with site-directed mutagenesis experiments to dissect the molecular basis of a unique allosteric property of crocodilian hemoglobin (Hb). Among vertebrate Hbs, crocodilian Hb is unique in that its O2-affinity is primarily modulated by bicarbonate ions rather than organic phosphates such as ATP. The mechanistic basis of this unique mode of allosteric regulatory control has remained a mystery. We report new findings regarding the specific substitutions responsible for the gain and loss of different allosteric interactions and the biophysical mechanisms by which they exert their functional effects.

ABSTRACT. Anti-fibrotic therapy remains an unmet medical need in human chronic liver diseases. Recently, Cytoglobin (CYGB) was reported to inhibit hepatic stellate cells (HSCs) activation and their collagen production. We aim to study anti-fibrotic property of 4 human globin-proteins including tetramer Hemoglobin (HB), monomer-Myoglobin (MB), -CYGB, and -Neuroglobin (NG). We produced recombinant human CYGB and NG. MB and HB were from commercials. IC50 values of ROS-scavenging activity of human HB, MB, CYGB and NG were measured. The bio-distribution of globin-proteins after in vitro and in vivo administration was traced. Cellular fractionation revealed that extracellularly added MB, NG, and CYGB, but not HB, penetrated human HSCs (HHSteCs) and located in membrane, cytoplasm and cytoskeletal fractions. Except HB, others scavenged reactive oxygen species generated spontaneously or stimulated by H2O2 or transforming growth factor β1 in HHSteCs and reduced collagen 1A1 production via suppressing its promoter activity. RNA-seq analysis of MB, NG and CYGB-treated HHSteCs revealed the common downregulation of extracellular matrix-encoding and fibrosis-related genes, and the upregulation of antioxidant genes or inactivated markers of HSCs including GATA, EST2, and PPARγ. Disruption of disulfide bond in NG decreased heme activity, superoxide-scavenging activity, and collagen inhibition capacity. Six weeks of CCl4 treatment in mice induces collagen deposition indicated by Sirius Red and Fast Green staining, αSMA expression, CD68 macrophage infiltration, and neutrophil populations. Intravenously injected MB, NGB, or CYGB at 1mg/kg BW, twice a week for last 4 weeks of CCl4, exhibited the clear attenuation of these manifestations. Conclusion: These findings revealed an unexpected and profound role for MB, NG and CYGB in maintaining HSCs in deactivated status and protect the mouse liver against cirrhosis, proposing the globin therapy as a new strategy to combat fibrotic liver disease.

ABSTRACT. Androglobin (Adgb), the youngest mammalian globin family member, owns its name to its predominant expression in testis. Adgb displays a unique chimeric domain structure among the globin family, consisting of an atypical permutated globin domain that is split into two parts by a calmodulin (CaM)-binding motif, an N-terminal calpain-like protease domain and a long C-terminal tail of yet unknown function. Here we will provide an overview of recent observations supporting a function of Adgb in ciliogenesis. Adgb is transcriptionally regulated by the master regulator of ciliogenesis FOXJ1, whereby its transcription not only relies on its promoter, but also on a distal enhancer region. Moreover, scRNAseq revealed expression of Adgb specifically in ciliated cells of the lungs, brain and female reproductive tract. Our data suggest a specific role for the isolated globin domain, likely resulting from auto-proteolytic cleavage, in the ciliary basis. Accordingly, Adgb KO mice develop a ciliopathy termed primary ciliary dyskinesia (PCD) including male infertility due to abnormal sperm flagellum formation, hydrocephalus, mucus accumulations in sinus and lungs, congenital heart defects, polycystic kidneys and rare cases of laterality defects. We found a ciliopathy-associated septin family member as specific Adgb interactor. In vitro data suggest that Adgb contributes to septin proteolysis in a CaM-dependent manner with most efficient CaM binding to Adgb upon isolation of the globin domain. Human PCD is also marked by airway oxidative stress and reduced nasal NO levels, which is commonly used as diagnostic approach, but with still unknown origin. Adgb thus represents an excellent candidate gene which may link ciliogenesis with airway ROS/RNS detoxification. Primary mouse tracheal epithelial cells from control and KO mice are currently exploited to further study the function of Adgb in ciliogenesis, and how this enigmatic globin interconnects oxygen physiology and ciliary function.

ABSTRACT. High-level expressed myoglobin (MB) is known to deliver oxygen in striated muscles. MB exists also at low levels in mammary epithelial cells, specifically in the lipid-secreting inner luminal cells of the milk duct epithelium where, however, MB’s function is unclear. Oxygenated MB is able to bind long-chain fatty acids (FAs) in vitro. Ablation of the MB gene in the heart and brown adipose tissue (reference below) switches substrate utilization from highly aerobic FA ß-oxidation to relatively O2-sparing glucose oxidation.

We now utilized mice and human breast cancer cells with targeted MB ablation to examine MB’s impact, and of its oxygenation status, on FA metabolism in mouse milk and mammary epithelia. MB expression shifted FA composition toward more saturated and shorter FA’s in milk and cells. Presence of MB also increased cytoplasmic FA solubility under normoxia and FA deposition to lipid droplets under severe hypoxia.

Since MB frequently occurs in mammary carcinoma, we further studied the globin´s role in breast cancer development and progression in vivo. We, thus, crossed PyMT and WapCreTRP53flox mouse models that both develop spontaneous breast cancer, yet differ in onset and tumor grade/type, with MB knock out mice. While the loss of MB in WapCre;Trp53flox mice had no effect on tumor development, it decreased tumor growth but increased tissue hypoxia and the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice.

Together, MB emerges as an intracellular O2-dependent shuttle of oxidizable FA substrates in normal and malignant breast epithelia. Regarding breast cancer, we propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification and outcome prediction. MB-targeted therapeutics in combination with standard strategy may offer a novel intervention concept for advanced metastatic breast cancer.

ABSTRACT. Photodynamic therapy (PDT) is a clinically approved methodology able to kill malignant cells and pathogens. Its efficacy relies on the cytotoxic effects of reactive oxygen species, in particular singlet oxygen, formed by excitation of a photoactivable molecule, called photosensitizer (PS), with visible light in presence of molecular oxygen. PDT is considered a promising alternative treatment, and its efficacy of the PDT against antibiotic-resistant bacteria has drawn a renewed attention. Essential properties that an effective photoactive system for PDT must possess, are high bioavalaibility of the PS, and targeting capability of the developed construct. Two important issues must be met. First, PSs are usually not water soluble molecules. Second, the lifetime of singlet oxygen is around 3.5 s, thus limiting its action radius to approximately 200 nm. Can heme-proteins be of help? Using proteins to solubilize PSs is an interesting but not widely exploited tool. We proposed Zinc substituted-myoglobin (ZnMb) as a promising system that combines a high quatum yield of production of singlet oxygen thanks to substitution of Fe(II) with Zn(II) at the center of porphyrinic ring (0.9), and the biocompatibility of the protein scaffold. The efficacy of this system has been proved on S. aureus with a decrease up to 99.99% in CFU. Due to the fact that ZnMb forms naturally during nitrate-free ham aging, this protein may represents a tool for Gram-positive bacteria decontamination of food. However, the efficacy of a construct for PDT is strongly dependent on its ability to target specific receptors on the plasma membrane of cancer cells or on the bacterial wall. To address this issue, we fused the gene of human myoglobin to a short peptide that targets a receptor overexpressed in some tumor cells. Preliminary experiments demonstrate that the chimeric construct binds prostate cancer cells, and the presence of the targeting agent is essential to obtain this interaction.