FEBS Letters

doi: 10.1002/1873-3468.14127pmid: N/A

Täuber, Susanne; Oliveri, Nancy F.; Kostakopoulou, Dora; Mahmoudi, Morteza

doi: 10.1002/1873-3468.14473pmid: 36052874

Santos, Tania C. B.; Dingjan, Tamir; Futerman, Anthony H.

doi: 10.1002/1873-3468.14457pmid: 35899376

Modern cell membranes contain a bewildering complexity of lipids, among them sphingolipids (SLs). Advances in mass spectrometry have led to the realization that the number and combinatorial complexity of lipids, including SLs, is much greater than previously appreciated. SLs are generated de novo by four enzymes, namely serine palmitoyltransferase, 3‐ketodihydrosphingosine reductase, ceramide synthase and dihydroceramide Δ4‐desaturase 1. Some of these enzymes depend on the availability of specific substrates and cofactors, which are themselves supplied by other complex metabolic pathways. The evolution of these four enzymes is poorly understood and likely depends on the co‐evolution of the metabolic pathways that supply the other essential reaction components. Here, we introduce the concept of the ‘anteome’, from the Latin ante (‘before’) to describe the network of metabolic (‘omic’) pathways that must have converged in order for these pathways to co‐evolve and permit SL synthesis. We also suggest that the current origin of life and evolutionary models lack appropriate experimental support to explain the appearance of this complex metabolic pathway and its anteome.

Pardella, Elisa; Ippolito, Luigi; Giannoni, Elisa; Chiarugi, Paola

doi: 10.1002/1873-3468.14441pmid: 35776088

Deregulated metabolism is a well‐known feature of several challenging diseases, including diabetes, obesity and cancer. Besides their important role as intracellular bioenergetic molecules, dietary nutrients and metabolic intermediates are released in the extracellular environment. As such, they may achieve unconventional roles as hormone‐like molecules by activating cell surface G‐protein‐coupled receptors (GPCRs) that regulate several pathophysiological processes. In this review, we provide an insight into the role of lactate, succinate, fatty acids, amino acids as well as ketogenesis‐derived and β‐oxidation‐derived intermediates as extracellular signalling molecules. Moreover, the mechanisms by which their cognate metabolite‐sensing GPCRs integrate nutritional and metabolic signals with specific intracellular pathways will be described. A better comprehension of these aspects is of fundamental importance to identify GPCRs as novel druggable targets.

Houghton, Fiona J.; Makhoul, Christian; Cho, Ellie Hyun‐Jung; Williamson, Nicholas A.; Gleeson, Paul A.

doi: 10.1002/1873-3468.14443pmid: 35789482

The small G protein Arl5b is localised on the trans‐Golgi network (TGN) and regulates endosomes‐to‐TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane‐bound Arl5b(Q70L)‐GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP‐Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN‐localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.

Katzy, Rebecca E.; Ferraz, Maria J.; Hazeu, Marc; Overkleeft, Hermen S.; Aerts, Johannes M. F. G.

doi: 10.1002/1873-3468.14448pmid: 35796054

Glycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing 13C atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope‐labelled precursor combined with liquid chromatography–mass spectrometry (MS)/MS analysis allows accurate determination of the IC50 values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells.

Teakel, Sarah; Marama, Michealla; Aragão, David; Tsimbalyuk, Sofiya; Mackie, Emily R. R.; Soares da Costa, Tatiana P.; Forwood, Jade K.; Cahill, Michael A.

doi: 10.1002/1873-3468.14471pmid: 35993565

We recently reported that the membrane‐associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b5 (cytb5) domain proteins, called cytb5M (MAPR‐like). Relative to classical cytb5 proteins, MAPR and ctyb5M proteins shared unique sequence elements and a distinct heme‐binding orientation at an approximately 90° rotation relative to classical cytb5, as demonstrated in the archetypal crystal structure of a cytb5M protein (PDB accession number 6NZX). Here, we present the crystal structure of an archaeal cytb5M domain (Methanococcoides burtonii WP_011499504.1, PDB:6VZ6). It exhibits similar heme binding to the 6NZX cytb5M, supporting the deduction that MAPR‐like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene.

Kägi, Jan; Makarchuk, Iryna; Wohlwend, Daniel; Melin, Frédéric; Friedrich, Thorsten; Hellwig, Petra

doi: 10.1002/1873-3468.14482pmid: 36029102

The reduction of oxygen to water is crucial to life and a central metabolic process. To fulfil this task, prokaryotes use among other enzymes cytochrome bd oxidases (Cyt bds) that also play an important role in bacterial virulence and antibiotic resistance. To fight microbial infections by pathogens, an in‐depth understanding of the enzyme mechanism is required. Here, we combine bioinformatics, mutagenesis, enzyme kinetics and FTIR spectroscopy to demonstrate that proton delivery to the active site contributes to the rate limiting steps in Cyt bd‐I and involves Asp58 of subunit CydB. Our findings reveal a previously unknown catalytic function of subunit CydB in the reaction of Cyt bd‐I.

White, Scott A.; Christofferson, Andrew J.; Grainger, Alastair I.; Day, Martin A.; Jarrom, David; Graziano, Antonio E.; Searle, Peter F.; Hyde, Eva I.

doi: 10.1002/1873-3468.14413pmid: 35648111

Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady‐state and stopped‐flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20–50 fold more active with NADPH than with NADH and that product release may be rate‐limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate‐binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half‐of‐sites mechanism.

Phillips, Robert S.; Anderson, Kaitlin L.; Gresham, Declan

doi: 10.1002/1873-3468.14469pmid: 35953460

d‐Glucosaminate‐6‐phosphate ammonia‐lyase (DGL) catalyzes the conversion of d‐glucosaminate‐6‐phosphate to 2‐keto‐3‐deoxyglutarate‐6‐phosphate, with stereospecific protonation of C‐3 of the product. The crystal structure of DGL showed that His‐163 could serve as the proton donor. H163A mutant DGL is fully active in the steady‐state reaction, and the pre‐steady‐state kinetics are very similar to those of wild‐type DGL. However, H163A DGL accumulates a transient intermediate with λmax at 293 nm during the reaction that is not seen with wild‐type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D2O shows that the product is a mixture of deuterated diastereomers at C‐3. These results establish that His‐163 is the proton donor in the reaction mechanism of DGL.