Generation and Export of Red Blood Cell ATP in Health and Disease

Metabolic homeostasis in animals depends critically on evolved mechanisms by which red blood cell (RBC) hemoglobin (Hb) senses oxygen (O2) need and responds accordingly. The entwined regulation of ATP production and antioxidant systems within the RBC also exploits Hb-based O2-sensitivity to respond to various physiologic and pathophysiologic stresses. O2 offloading, for example, promotes glycolysis in order to generate both 2,3-DPG (a negative allosteric effector of Hb O2 binding) and ATP. Alternatively, generation of the nicotinamide adenine dinucleotide phosphate (NADPH) critical for reducing systems is favored under the oxidizing conditions of O2 abundance. Dynamic control of ATP not only ensures the functional activity of ion pumps and cellular flexibility, but also contributes to the availability of vasoregulatory ATP that can be exported when necessary, for example in hypoxia or upon RBC deformation in microvessels. RBC ATP export in response to hypoxia or deformation dilates blood vessels in order to promote efficient O2 delivery. The ability of RBCs to adapt to the metabolic environment via differential control of these metabolites is impaired in the face of enzymopathies [pyruvate kinase deficiency; glucose-6-phosphate dehydrogenase (G6PD) deficiency], blood banking, diabetes mellitus, COVID-19 or sepsis, and sickle cell disease. The emerging availability of therapies capable of augmenting RBC ATP, including newly established uses of allosteric effectors and metabolite-specific additive solutions for RBC transfusates, raises the prospect of clinical interventions to optimize or correct RBC function via these metabolite delivery mechanisms.

A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme

Main conclusion A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Abstract Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.

State-Selective Frustration as a Key Driver of Allosteric Pluripotency

Allosteric pluripotency arises when an allosteric effector switches from agonist to antagonist depending on the experimental conditions. For example, the Rp-cAMPS ligand of Protein Kinase A (PKA) switches from agonist...

Cholesterol binding to the sterol-sensing region of Niemann Pick C1 protein confines dynamics of its N-terminal domain

Lysosomal accumulation of cholesterol is a hallmark of Niemann Pick type C (NPC) disease caused by mutations primarily in the lysosomal membrane protein NPC1. NPC1 contains a transmembrane sterol sensing domain (SSD), which is supposed to regulate protein activity upon cholesterol binding, but the mechanisms underlying this process are poorly understood. Using atomistic simulations, we show that the binding of cholesterol to the SSD of NPC1 suppresses conformational dynamics of the luminal domains which otherwise bring the luminal N-terminal domain (NTD) closer to the lipid bilayer. The presence of an additional 20% membrane cholesterol has negligible impact on this process. We propose that cholesterol acts as an allosteric effector, and the modulation of NTD dynamics by the SSD-bound cholesterol constitutes an allosteric feedback mechanism in NPC1 which controls cholesterol abundance in the lysosomal membrane.

Oxygenation properties and underlying molecular mechanisms of hemoglobins in plateau zokor (Eospalax baileyi)

The plateau zokor ( Eospalax baileyi) is a species of subterranean rodent endemic to the Tibetan Plateau. It is well adapted to the cold and hypoxic and hypercapnic burrow. To study the oxygenation properties of plateau zokor hemoglobins (Hbs), we measured intrinsic Hb-O2 affinities and their sensitivities to pH (Bohr effect); CO2; Cl−, 2,3-diphosphoglycerate (DPG); and temperature using purified Hbs from zokor and mouse. The optimal deoxyHb model of plateau zokor was constructed and used to study its structural characteristics by molecular dynamics simulations. O2 binding results revealed that plateau zokor Hbs exhibit remarkably high intrinsic Hb-O2 affinity, low CO2 effects compared with human and the relatively low anion allosteric effector sensitivities (DPG and Cl−) at normal temperature, which would safeguard the pulmonary Hb-O2 loading under hypoxic and hypercapnic conditions. Furthermore, the high anion allosteric effector sensitivities at low temperature and low temperature sensitivities of plateau zokor Hbs would facilitate the releasing of O2 in cold extremities and metabolic tissues. However, the high Hb-O2 affinity of plateau zokor is not compensated by high pH sensitivity as the Bohr factors of plateau zokor Hbs were as low as those of mouse. The results of molecular dynamics simulations revealed the reduced hydrogen bonding between the α1β1- and α2β2-dimer interface of deoxyHb in zokor compared with mouse. It may be the primary mechanism of the high intrinsic Hb-O2 affinities in zokor. Specifically, substitution of the 131Ser→Asn in the α2-chain weakened the connection between α1- and β2-subunit.

Functional Properties of Hb S and Hb C in Stored Cpda-1 Red Blood Cells Concentrate — a Prospective Study

Most of the 1,300 human hemoglobin (Hb) variants are characterized by normal O2 binding, including here the highly prevalent Hbs S [HBB:c.20A>T; p.Glu6Val] and C [HBB:c.19G>A; p.Glu6Lys]. Hb S and Hb C carriers are generally asymptomatic and might be admitted as blood donors. However, the functional efficiency of these variants in standard blood bags - red blood cells (RBC) concentrates - under the interference of anticoagulant and storage is not well established. In this prospective study, three samples of RBC concentrates (phenotypes AA, AS and AC), with citrate phosphate dextrose adenine (CPDA-1) as anticoagulant, were analyzed for equilibrium tests and compared to fresh Hb A without anticoagulant. The Hb samples were extracted after 48 hours and 35 days of storage and purified by exclusion and ion exchange chromatography (Sephadex G-25 and Amberlite MB-3 columns in Hepes buffer without NaCl). O2 affinity was evaluated by spectrophotometry-tonometry method, by determining the p50 in stripped lysates ([Hb]=70 μM/Heme), in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) in all studied pHs. The activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. The p50 of Hb A after 35 days in stored blood bag was decreased in all pHs, in both presence and absence of IHP, when compared to fresh Hb A, suggesting that the anticoagulant and the storage might affect HbA-O2 binding, increasing its affinity for the ligand. No other differences were observed: heme-heme cooperativity and Bohr effect were similar to fresh Hb A without anticoagulant. The same was found when stored Hb AS was compared to Hb A from blood bag (35 days storage, in the stripped state and under the interference of IHP). However, the stripped lysate containing Hb AC revealed an important reduction of the O2 affinity after both 48 hours and 35 days of storage, mainly at pHs ³ 7, thus interfering in the Bohr effect. The heme-heme cooperativity resulted in normal n coefficients, indicating that the Hb-O2 ligation remained cooperative, in spite of the significant reduction of O2 affinity. Similar results were observed in the presence of IHP (48 hours and 35 days), although Bohr effect was reestablished, indicating that the supplementation with a 2,3-BPG-like allosteric effector might restore some Hb-ion interactions, and therefore, Hb C function. As the 2,3-BPG is consumed with storage of RBC concentrates, Hb AC showed an even lower affinity for O2 after 35 days of storage. Although still preliminary, our results indicate a significant functional reduction of Hb C in RBC concentrates in standard blood bag (CPDA-1 anticoagulant), proportional to the time of storage. Considering that the screening for hemoglobinopathies in the blood banks routine is mainly focused in the detection of Hb S, a greater understanding about the properties of native Hb and its most frequent variants in stored RBC concentrates could also contribute with the determination of safe parameters in hemotherapy based on the functional efficiency of each hemoglobin. Financial Support: Fapesp, CNPq, Capes, Faepex-Unicamp. Disclosures No relevant conflicts of interest to declare.