Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations

Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions. Under hypoxia, cytoglobin generates nitric oxide, which is strongly modulated by the oxidation state of the cysteines. This gives a plausible role for this biochemistry in controlling nitric oxide homeostasis. Mutations to control specific properties of hemoglobin and myoglobin, including nitric oxide binding/scavenging and the nitrite reductase activity of various globins, have been reported. We have mapped these key mutations onto cytoglobin, which represents the E7 distal ligand, B2/E9 disulfide, and B10 heme pocket residues, and examined the nitric oxide binding, nitric oxide dioxygenase activity, and nitrite reductase activity. The Leu46Trp mutation decreases the nitric oxide dioxygenase activity &gt; 10,000-fold over wild type, an effect 1000 times greater than similar mutations with other globins. By understanding how particular mutations can affect specific reactivities, these mutations may be used to target specific cytoglobin activities in cell or animal models to help understand the precise role(s) of cytoglobin under physiological and pathophysiological conditions.</jats:p

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions

VarLOCK: Sequencing-Independent, Rapid Detection of SARS-CoV-2 Variants of Concern for Point-of-Care Testing, qPCR Pipelines and National Wastewater Surveillance

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs

VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

The COVID-19 pandemic continues to pose a threat to the general population. The ongoing vaccination programs provide protection to individuals and facilitate the opening of society and a return to normality. However, emergent and existing SARS-CoV-2 variants capable of evading the immune system endanger the efficacy of the vaccination strategy. To preserve the efficacy of SARS-CoV-2 vaccination globally, aggressive and effective surveillance for known and emerging SARS-CoV-2 Variants of Concern (VOC) is required. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. In this work, we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to allow rapid and sensitive discrimination of VOCs, that can be used at point of care and/or implemented in the pipelines of small or large testing facilities, and even determine proportion of VOCs in pooled population-level wastewater samples. This technology aims to complement the ongoing sequencing efforts to allow facile and, crucially, rapid identification of individuals infected with VOCs to help break infection chains. Here, we show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants. In addition, we show the rapid adaptability of the technique for new and emerging VOCs such as Omicron

Strong modulation of nitrite reductase activity of cytoglobin by disulfide bond oxidation: Implications for nitric oxide homeostasis

Globin-mediated nitric oxide (NO) dioxygenase and nitrite reductase activities have been proposed to serve protective functions within the cell by scavenging or generating NO respectively. Cytoglobin has rapid NO dioxygenase activity, similar to other globins, however, the apparent rates of nitrite reductase activity have been reported as slow or negligible. Here we report that the activity of cytoglobin nitrite reductase activity is strongly dependent on the oxidation state of the two surface-exposed cysteine residues. The formation of an intramolecular disulfide bond between cysteines C38 and C83 enhances the nitrite reductase activity by 50-fold over that of the monomer with free sulfhydryl or 140-fold over that of the dimer with intermolecular disulfide bonds. The NO dioxygenase reactivity of cytoglobin is very rapid with or without disulfide bond, however, binding of the distal histidine following dissociation of the nitrate are affected by the presence or absence of the disulfide bond. The nitrite reductase activity reported here for the monomer with intramolecular disulfide is much higher than of those previously reported for other mammalian globins, suggesting a plausible role for this biochemistry in controlling NO homeostasis the cell under oxidative and ischemic conditions

Studies of the effect of cysteine and heme pocket mutations of the nitric oxide dioxygenase and nitrite reductase activities of human Cytoglobin and Androglobin heme domain

Oxygen delivery to hypoxic tissues along with the interplay between hemoglobins and hypoxic tissues have been an area of interest, the nitrite reductase activity (NiR) and the nitric oxide dioxygenase activity (NOD) working in tandem is a proposed regulatory response which enhances oxygen levels in hypoxic tissues, serving to protect tissues against hypoxia. In hypoxic conditions, NO is generated via the NiR activity, while in normoxic condition NO is consumed via the NOD activity. With NO being a versatile signalling molecule and a potent relaxant of the vasculature, NO homeostasis by the NiR and NOD activity regulates vascular tone in response to oxygen demand. In traditional oxygen carriers like hemoglobin (Hb) and myoglobin (Mb), NO homeostasis via the NOD and NiR activities is seen as an additional function and is well studied and characterised in these globins. Scientific inquiry on hemoglobin superfamily and advances in genomics have led to the discovery of new hemoglobins, including an expanded human hemoglobin family with the discovery of additional members like Neuroglobin (Ngb) Cytoglobin (Cygb) and Androglobin (Adgb). These fresh additions are hexacoordinate, unlike Mb and Hb which have their sixth coordination site vacant in the absence of external ligands, Cygb and Adgb have their sixth coordination site occupied, by a histidine and glutamine respectively. Cygb is a low abundance vertebrate globin of ubiquitous expression, distantly related to Mb and upregulated during cellular hypoxia. Crystallographic data places Cygb as a monomer with free sulfhydryl and dimer with an intermolecular bond between two monomeric subunits. Biochemical studies have observed Cygb also existing as a monomer with an intramolecular disulfide bond, this form of the protein lacks crystallographic data. Adgb is a chimeric globin with a rearranged globin domain, Adgb lacks biochemical characterisation with little information obtained about its biochemical properties. However, Adgb highly is expressed in the testes and gene expression upregulated during spermatogenesis. Despite efforts to probe the biochemical activities of these additional members, their physiological roles are still not clearly defined. In this study, we report that the formation of an intramolecular disulfide bond between cysteines C38 and C83 enhances the ii nitrite reductase activity by 50-fold over that of the monomer with free sulfhydryl or 140-fold over that of the dimer with intermolecular disulfide bonds. The NO dioxygenase reactivity of cytoglobin is also very rapid with or without disulfide bond. We have targeted amino acid residues (H81A, L46F, L46W, C38,83S) in Cygb by mapping mutations targeting specific properties of hemoglobin (Hb) and myoglobin (Mb), including O2 binding and NO binding/scavenging as well as the NiR activity of Ngb, in addition to other rationalised mutations (C38,83S). Mutations of amino acid residues at the distal pocket successfully enhanced or attenuated the NiR and NOD of Cygb, by understanding how particular mutations can affect specific reactivities, these mutations may be used to target the NiR or NOD (e.g. by CRISPR/Cas9) in cell or animal models to help understand the precise role (or roles) of Cygb under physiological and pathophysiological conditions. Ultrafast and laser flash photolysis aided probing of the roles of the cysteines on ligand migration pathway, data suggests the cysteines play a role in ligand entry and exit from Cygb. We have re-evaluated the structural sequence assigned to the globin domain of Adgb (Adgb-GD), and we propose that part of the original structural sequence alignment was in error. Upon correction, we have expressed the Adgb-GD as a stable protein at neutral or alkali pH. Additionally, with a stable form of the protein expressed, we have identified a unique feature of the Adgb-GD. Adgb-GD binds nitric oxide in 5-coordinate heme configuration, unlike any other human globin, but similar to that observed with guanylate cyclase, cytochrome c’. Adgb-GD displays a high nitrite reductase activity, which is influenced by the redox state of the disulfide bond. Spectra characterisation depicts Adgb-GD as a hexacoordinate globin, comparative spectra analysis with Cytochrome C’ suggest Adgb-GD may possess similar ligand binding properties

The circularly permuted globin domain of Androglobin exhibits atypical heme stabilization and nitric oxide interaction

International audienceIn the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin’s missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions