Carbonic anhydrase V (CA V), a mitochondrial enzyme, was first isolated from guinea pig liver and subsequently identified in mice and humans. Later, studies revealed that the mouse genome contains two mitochondrial CA sequences, named Car5A and Car5B. The CA VA enzyme is most highly expressed in the liver, whereas CA VB shows a broad tissue distribution. Car5A knockout mice showed a predominant role for CA VA in ammonia detoxification, while the roles of CA VB in ureagenesis and gluconeogenesis were evident only in the absence of CA VA. Previous studies have suggested that CA VA is mainly involved in the provision of HCO for biosynthetic processes. In children, mutations in the CA5A gene led to reduced CA activity, and the enzyme was sensitive to increased temperature. The metabolite profiles of these children showed a reduced supply of HCO to the enzymes that take part in intermediary metabolism: carbamoylphosphate synthetase, pyruvate carboxylase, propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase. Although the role of CA VB is still poorly understood, a recent study reported that it plays an essential role in human Sertoli cells (hSCs), which sustain spermatogenesis. Metabolic disease associated with CA VA appears to be more common than other inborn errors of metabolism (IEM) and responds well to treatment with N-carbamyl-l-glutamate (NCG). Therefore, early identification of hyperammonemia will allow specific treatment with NCG and prevent neurologic sequelae. CA VA deficiency should therefore be considered a treatable condition in the differential diagnosis of hyperammonemia in neonates and young children. Abstract figure legend Carbonic anhydrase isozymes VA and VB represent mitochondrial enzymes which contribute to several physiological functions mainly in intermediary metabolism. The liver hepatocytes are the main source of carbonic anhydrase VA with weaker signals in brain, testis, and muscle. The VB isozyme is more widely spread in several organs, such as brain, heart, liver, lung, kidney, spleen, intestine, testis, muscle, and pancreas. This article is protected by copyright. All rights reserved.

Prothrombin, protein C, and factors VII, IX, and X are vitamin K (VK)-dependent coagulation proteins that play an important role in the initiation, amplification, and subsequent attenuation of the coagulation response. Blood coagulation evolved in the common vertebrate ancestor as a specialization of the complement system and immune response, which in turn bear close evolutionary ties with developmental enzyme cascades. There is currently no comprehensive analysis of the evolutionary changes experienced by these coagulation proteins during the radiation of vertebrates and little is known about conservation of residues that are important for zymogen activation and catalysis.

Hutchinson-Gilford progeria syndrome is a premature aging disease caused by LMNA gene mutation and the production of a truncated prelamin A protein "progerin" that elicits cellular and organismal toxicity. Progerin accumulates in the vasculature, being especially detrimental for vascular smooth muscle cells (VSMC). Vessel stiffening and aortic atherosclerosis in HGPS patients are accompanied by VSMC depletion in the medial layer, altered extracellular matrix (ECM), and thickening of the adventitial layer. Mechanisms whereby progerin causes massive VSMC loss and vessel alterations remain poorly understood. Mature VSMC retain phenotypic plasticity and can switch to a synthetic/proliferative phenotype. Here, we show that progerin expression in human and mouse VSMC causes a switch towards the synthetic phenotype. This switch elicits some level of replication stress in normal cells, which is exacerbated in the presence of progerin, leading to telomere fragility, genomic instability, and ultimately VSMC death. Calcitriol prevents replication stress, telomere fragility, and genomic instability, reducing VSMC death. In addition, RNA-seq analysis shows induction of a profibrotic and pro-inflammatory aging-associated secretory phenotype upon progerin expression in human primary VSMC. Our data suggest that phenotypic switch-induced replication stress might be an underlying cause of VSMC loss in progeria, which together with loss of contractile features and gain of profibrotic and pro-inflammatory signatures contribute to vascular stiffness in HGPS.

Allostery is a property of biological macromolecules featuring cooperative ligand binding and regulation of ligand affinity by effectors. The definition was introduced by Monod and Jacob in 1963, and formally developed as the "concerted model" by Monod, Wyman, and Changeux in 1965. Since its inception, this model of cooperativity was seen as distinct from and not reducible to the "sequential model" originally formulated by Pauling in 1935, which was developed further by Koshland, Nemethy, and Filmer in 1966. However, it is difficult to decide which model is more appropriate from equilibrium or kinetics measurements alone. In this paper, we examine several cooperative proteins whose functional behavior, whether sequential or concerted, is established, and offer a combined approach based on functional and structural analysis. We find that isologous, mostly helical interfaces are common in cooperative proteins regardless of their mechanism. On the other hand, the relative contribution of tertiary and quaternary structural changes, as well as the asymmetry in the liganded state, may help distinguish between the two mechanisms.

A State of the Art lecture titled "Cryo-EM structures of coagulation factors" was presented at the ISTH Congress in 2022. Cryogenic electron microscopy (cryo-EM) is a revolutionary technique capable of solving the structure of high molecular weight proteins and their complexes, unlike nuclear magnetic resonance (NMR), and under conditions not biased by crystal contacts, unlike X-ray crystallography. These features are particularly relevant to the analysis of coagulation factors that are too big for NMR and often recalcitrant to X-ray investigation. Using cryo-EM, we have solved the structures of coagulation factors V and Va, prothrombinase on nanodiscs, and the prothrombin-prothrombinase complex. These structures have advanced basic knowledge in the field of thrombosis and hemostasis, especially on the function of factor V and the molecular mechanism for prothrombin activation, and set the stage for exciting new lines of investigation. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Rift Valley fever virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the order Bunyavirales, whose replication depends on the enzymatic activity of the viral L protein. Screening for RVFV inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 47 novel RVFV inhibitors with selective indexes from 1.1-103 and 50% effective concentrations of 1.2-56 μM in Vero cells, primarily α-Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against a second Bunyavirus, La Crosse Virus (LACV), and the flavivirus Zika (ZIKV). These data indicate that the α-Hydroxytropolone and N-Hydroxypyridinedione chemotypes should be investigated in the future to determine their mechanism(s) of action allowing further development as therapeutics for RVFV and LACV, and these chemotypes should be evaluated for activity against related pathogens, including Hantaan virus, severe fever with thrombocytopenia syndrome virus, Crimean-Congo hemorrhagic fever virus.

Dysregulated lipid metabolism is common in infection and inflammation and is a part of the complex milieu underlying the pathophysiological sequelae of disease. Sepsis is a major cause of mortality and morbidity in the world and is characterized by an exaggerated host response to an infection. Metabolic changes, including alterations in lipid metabolism, likely are important in sepsis pathophysiology. Here, we designed an cell culture model using endothelial cells, , and neutrophils to mimic sepsis in a simplified cell model. Lipid alterations were studied in the presence of the pathogenic strain CFT073 and non-pathogenic strain JM109. We employed untargeted lipidomics to first identify lipid changes and then targeted lipidomics to confirm changes. Both unique and shared lipid signatures were identified in cocultures with these strains. In the absence of neutrophils, the CFT073 strain elicited alterations in lysophosphatidylcholine and diglyceride molecular species during coculture while both strains led to increases in phosphatidylglycerols. Lipid alterations in these cocultures changed with the addition of neutrophils. In the presence of neutrophils with and endothelial cells, triglyceride increases were a unique response to the CFT073 strain while phosphatidylglycerol and diglyceride increases occurred in response to both strains. Phosphatidylethanolamine also increased in neutrophils, and endothelial cells cocultures, and this response was greater in the presence of the CFT073 strain. We further evaluated changes in phosphatidylethanolamine in a rat model of sepsis, which showed multiple plasma phosphatidylethanolamine molecular species were elevated shortly after the induction of sepsis. Collectively, these findings demonstrate unique lipid responses by co-cultures of with endothelial cells which are dependent on the strain as well as the presence of neutrophils. Furthermore, increases in phosphatidylethanolamine levels in CFT073 urosepsis , endothelial cell, neutrophil cocultures were similarly observed in the plasma of septic rats.