HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

Biochemical and morphological modifications in rabbit Achilles tendon during maturation and ageing.

1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow.     

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.     

Recognition of Smac-mimetic compounds by the BIR domain of cIAP1

Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.     

Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography

Lipopolysaccharide (LPS) biosynthesis represents an underexploited target pathway for novel antimicrobial development to combat the emergence of multidrug‐resistant bacteria. A key player in LPS synthesis is the enzyme D ‐arabinose‐5‐phosphate isomerase (API), which catalyzes the reversible isomerization of D ‐ribulose‐5‐phosphate to D ‐arabinose‐5‐phosphate, a precursor of 3‐deoxy‐D ‐manno‐octulosonate that is an essential residue of the LPS inner core. API is composed of two main domains: an N‐terminal sugar isomerase domain (SIS) and a pair of cystathionine‐β‐synthase domains of unknown function. As the three‐dimensional structure of an enzyme is a prerequisite for the rational development of novel inhibitors, we present here the crystal structure of the SIS domain of a catalytic mutant (K59A) of E. coli D ‐arabinose‐5‐phosphate isomerase at 2.6‐Å resolution. Our structural analyses and comparisons made with other SIS domains highlight several potentially important active site residues. In particular, the crystal structure allowed us to identify a previously unpredicted His residue (H88) located at the mouth of the active site cavity as a possible catalytic residue. On the basis of such structural data, subsequently supported by biochemical and mutational experiments, we confirm the catalytic role of H88, which appears to be a generally conserved residue among two‐domain isomerases.     

Linking genotype to phenotype in a changing ocean: inferring the genomic architecture of a blue mussel stress response with genome‐wide association

A key component to understanding the evolutionary response to a changing climate is linking underlying genetic variation to phenotypic variation in stress response. Here, we use a genome‐wide association approach (GWAS) to understand the genetic architecture of calcification rates under simulated climate stress. We take advantage of the genomic gradient across the blue mussel hybrid zone (Mytilus edulis and Mytilus trossulus) in the Gulf of Maine (GOM) to link genetic variation with variance in calcification rates in response to simulated climate change. Falling calcium carbonate saturation states are predicted to negatively impact many marine organisms that build calcium carbonate shells – like blue mussels. We sampled wild mussels and measured net calcification phenotypes after exposing mussels to a ‘climate change’ common garden, where we raised temperature by 3°C, decreased pH by 0.2 units and limited food supply by filtering out planktonic particles >5 μm, compared to ambient GOM conditions in the summer. This climate change exposure greatly increased phenotypic variation in net calcification rates compared to ambient conditions. We then used regression models to link the phenotypic variation with over 170 000 single nucleotide polymorphism loci (SNPs) generated by genotype by sequencing to identify genomic locations associated with calcification phenotype, and estimate heritability and architecture of the trait. We identified at least one of potentially 2–10 genomic regions responsible for 30% of the phenotypic variation in calcification rates that are potential targets of natural selection by climate change. Our simulations suggest a power of 13.7% with our study's average effective sample size of 118 individuals and rare alleles, but a power of >90% when effective sample size is 900.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.