2-oxoglutarate-dependent dioxygenases drive expansion of steroidal alkaloid structural diversity in the genus Solanum.

Solanum steroidal glycoalkaloids (SGAs) are renowned defence metabolites exhibiting spectacular structural diversity. Genes and enzymes generating the SGA precursor pathway, SGA scaffold and glycosylated forms have been largely identified. Yet, the majority of downstream metabolic steps creating the vast repertoire of SGAs remain untapped. Here, we discovered that members of the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (2-ODD) family play a prominent role in SGA metabolism, carrying out three distinct backbone-modifying oxidative steps in addition to the three formerly reported pathway reactions. The GLYCOALKALOID METABOLISM34 (GAME34) enzyme catalyses the conversion of core SGAs to habrochaitosides in wild tomato S. habrochaites. Cultivated tomato plants overexpressing GAME34 ectopically accumulate habrochaitosides. These habrochaitoside enriched plants extracts potently inhibit Puccinia spp. spore germination, a significant Solanaceae crops fungal pathogen. Another 2-ODD enzyme, GAME33, acts as a desaturase (via hydroxylation and E/F ring rearrangement) forming unique, yet unreported SGAs. Conversion of bitter α-tomatine to ripe fruit, nonbitter SGAs (e.g. esculeoside A) requires two hydroxylations; while the known GAME31 2-ODD enzyme catalyses hydroxytomatine formation, we find that GAME40 catalyses the penultimate step in the pathway and generates acetoxy-hydroxytomatine towards esculeosides accumulation. Our results highlight the significant contribution of 2-ODD enzymes to the remarkable structural diversity found in plant steroidal specialized metabolism.

Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Magnetization Transfer to Enhance NOE Cross-Peaks among Labile Protons: Applications to Imino-Imino Sequential Walks in SARS-CoV-2-Derived RNAs.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.

Polyamines Mediate Folding of Primordial Hyperacidic Helical Proteins.

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.

D-Glutamate production by stressed Escherichia coli gives a clue for the hypothetical induction mechanism of the ALS disease.

In the search for the origin of Amyotrophic Lateral Sclerosis disease (ALS), we hypothesized earlier (Monselise, 2019) that D-amino acids produced by stressed microbiome may serve as inducers of the disease development. Many examples of D-amino acid accumulation under various stress conditions were demonstrated in prokaryotic and eukaryotic cells. In this work, wild-type Escherichia coli, members of the digestive system, were subjected to carbon and nitrogen starvation stress. Using NMR and LC-MS techniques, we found for the first time that D-glutamate accumulated in the stressed bacteria but not in control cells. These results together with the existing knowledge, allow us to suggest a new insight into the pathway of ALS development: D-glutamate, produced by the stressed microbiome, induces neurobiochemical miscommunication setting on C1q of the complement system. Proving this insight may have great importance in preventive medicine of such MND modern-age diseases as ALS, Alzheimer, and Parkinson.

The chemokines CCL5 and CXCL12 exhibit high-affinity binding to N-terminal peptides of the non-cognate receptors CXCR4 and CCR5, respectively.

CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors. CCL5 contains one conserved binding site for a sulfated tyrosine residue, whereas CXCL12 is unique in having two additional sites for sulfated/nonsulfated tyrosine residues. In this study, N-terminal (Nt) CXCR4 peptides were found to bind CCL5 with somewhat higher affinities in comparison to those of short Nt-CCR5(8-20) peptides with the same number of sulfated tyrosine residues. Similarly, a long Nt-CCR5(1-27)(s Y3,s Y10,s Y14) peptide cross reacts with CXCL12 and with lower KD in comparison to its binding to CCL5. Intermolecular nuclear overhauser effect (NOE) measurements were used to decipher the mechanism of the chemokine/Nt-receptor peptide binding. The Nt-CXCR4 peptides interact with the conserved CCL5 tyrosine sulfate-binding site by an allovalency mechanism like that observed for CCL5 binding of Nt-CCR5 peptides. Nt-CCR5 peptides bind CXCL12 in multiple modes analogous to their binding to HIV-1 gp120 and interact with all three tyrosine/sulfated tyrosine-binding pockets of CXCL12. We suggest that the chemokine-receptors Nt-segments bind promiscuously to cognate and non-cognate chemokines and in a mechanism that is dependent on the number of binding pockets for tyrosine residues found on the chemokine. In conclusion, common features shared among the chemokine-receptors' Nt-segments such as multiple tyrosine residues that are potentially sulfated, and a large number of negatively charged residues are the reason of the cross binding observed in this study.

Glucagon-like peptide-1 analogs activate AMP kinase leading to reversal of the Warburg metabolic switch in breast cancer cells.

Breast cancer (BC) is associated with type 2 diabetes mellitus (T2DM) and obesity. Glucagon-like peptide (GLP)-1 regulates post-prandial insulin secretion, satiety, and gastric emptying. Several GLP-1 analogs have been FDA-approved for the treatment of T2DM and obesity. Moreover, GLP-1 regulates various metabolic activities across different tissues by activating metabolic signaling pathways like adenosine monophosphate (AMP) activated protein kinase (AMPK), and AKT. Rewiring metabolic pathways is a recognized hallmark of cancer, regulated by several cancer-related pathways, including AKT and AMPK. As GLP-1 regulates AKT and AMPK, we hypothesized that it alters BC cells' metabolism, thus inhibiting proliferation. The effect of the GLP-1 analogs exendin-4 (Ex4) and liraglutide on viability, AMPK signaling and metabolism of BC cell lines were assessed. Viability of BC cells was evaluated using colony formation and MTT/XTT assays. Activation of AMPK and related signaling effects were evaluated using western blot. Metabolism effects were measured for glucose, lactate and ATP. Exendin-4 and liraglutide activated AMPK in a cAMP-dependent manner. Blocking Ex4-induced activation of AMPK by inhibition of AMPK restored cell viability. Interestingly, Ex4 and liraglutide reduced the levels of glycolytic metabolites and decreased ATP production, suggesting that GLP-1 analogs impair glycolysis. Notably, inhibiting AMPK reversed the decline in ATP levels, highlighting the role of AMPK in this process. These results establish a novel signaling pathway for GLP-1 in BC cells through cAMP and AMPK modulation affecting proliferation and metabolism. This study suggests that GLP-1 analogs should be considered for diabetic patients with BC.