Residual helicity at the active site of the histidine phosphocarrier, HPr, modulates binding affinity to its natural partners

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first a-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first a-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. The refore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

A phosphorylation-induced switch in the nuclear localization sequence of the intrinsically disordered nupr1 hampers binding to importin

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin a, of which there are several human isoforms; among them, importin a3 (Impa3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impa3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impa3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impa3 with low micromolar affinity (1.7–27 µM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i, i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impa3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure

A translational approach to assess the metabolomic impact of stabilized gold nanoparticles by NMR spectroscopy

[EN] Gold nanoparticles have high potential in the biomedical area, especially in disease diagnosis and treatment. The application of these nanoparticles requires the presence of stabilizers to avoid their agglomeration. Nowadays, there is a lack of reliable methods for characterising the effect of stabilised nanoparticles on biological systems. To this end, in this study, we apply an experimental approach based on nuclear magnetic resonance spectroscopy to study the effect of gold nanoparticles, stabilised with cerium oxide or chitosan, on a human cancer cell model. The results showed that both systems have a significant effect, even at non-toxic levels, on the cellular antioxidant system. However, although particles functionalised with chitosan exerted a strong effect on the aerobic respiration, nanoparticles stabilised with cerium oxide had a higher impact on the mechanisms associated with anaerobic energy production. Therefore, even though both systems contained similar gold nanoparticles, the presence of different stabilizers strongly influenced their mode of action and potential applications in biomedicine.This work was supported by the Carlos III Health Institute, the European Regional Development Fund (PI16/02064 and CP13/00252) and the Spanish Ministerio de Economia y Competitividad (SAF2014-53977-R, SAF2017-89229-R and RD12/0036/0025). In addition, JRH is a recipient of a contract from the Ministry of Health of the Carlos III Health Institute.Herance, JR.; García Gómez, H.; Guitierrez Carcedo, P.; Navalón Oltra, S.; Pineda-Lucena, A.; Palomino-Schätzlein, M. (2019). A translational approach to assess the metabolomic impact of stabilized gold nanoparticles by NMR spectroscopy. The Analyst. 144(4):1265-1274. https://doi.org/10.1039/c8an01827hS126512741444Shi, J., Kantoff, P. W., Wooster, R., & Farokhzad, O. C. (2016). Cancer nanomedicine: progress, challenges and opportunities. 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Metabolic Fingerprint of Acromegaly and its Potential Usefulness in Clinical Practice

Insulin-like growth factor-1 (IGF-1) and growth hormone (GH) levels are the main targets for monitoring acromegaly activity, but they are not in close relationship with the clinical course of the disease and the associated comorbidities. The present study was aimed at identifying metabolites that could be used as biomarkers for a better disease phenotyping. For this purpose, metabolic fingerprint using an untargeted metabolomic approach was examined in serum from 30 patients with acromegaly and 30 age-matched controls. Patients with acromegaly presented fewer branched-chain amino acids (BCAAs) compared to the control group (valine: 4.75 ± 0.87 vs. 5.20 ± 1.06 arbitrary units (AUs), p < 0.05; isoleucine: 2.54 ± 0.41 vs. 2.80 ± 0.51 AUs; p < 0.05). BCAAs were also lower in patients with active disease compared to patients with normal levels of IGF-1 with or without medical treatment. GH, but not IGF-1, serum levels were inversely correlated with both valine and isoleucine. These findings indicate that low levels of BCAAs represent the main metabolic fingerprint of acromegaly and that GH, rather than IGF-1, might be the primary mediator. In addition, our results suggest that the assessment of BCAAs could help to identify active disease and to monitor the response to therapeutic strategies

Human milk and mucosal lacto- and galacto-N-biose synthesis by transgalactosylation and their prebiotic potential in Lactobacillus species

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-D-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants

Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α

13 pags., 7 figs.Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R, R, R and η), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.This work was supported bySpanish Ministry of Economy and Competitiveness [CTQ2015-64445-R (to JLN) ]with European ERDF funds;by the French La Ligue Contre le Cancer, INCa, Canceropole PACA, DGOS (la bellisation SIRIC) and INSERM (to JLI). The NMR equipment used in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OPERDF of Comunitat Valenciana 2014-2020). BR acknowledges kind hospitality in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy

Residual helicity at the active site of the histidine phosphocarrier, HPr, modulates binding affinity to its natural partners

18 pags., 4 figs., 5 tabs. -- This article belongs to the Special Issue Folding and Design of α-Helical Proteins and Peptides: Theory Meets Nanomaterials, Biotechnology and HealthThe phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr is mainly disordered. We attempted to improve the affinity of HPr to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. The refore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI2018-097991-B-I00 to J.L.N., BFU2016-78232-P to A.V.-C., BES-2017-080739 to D.O.-A., and RTI2018-101969-J-I00 to A.F.). The NMR equipment used in this work was funded by Generalitat Valenciana (Spain) and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana (Spain) 2014–2020)

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity

Antimicrobial peptides and their superior fluorinated analogues: structure-activity relationships as revealed by NMR spectroscopy and MD calculations

9 pag., 6 fig, 3 tab.The conformations of two synthetic pentapeptides with antimicrobial activity and their 4-fluorophenylalanine (Pff)-containing analogues (ArXArXAr-NH2; Ar=Phe, Pff; X=Lys, Arg) have been studied. NMR experiments carried out both in aqueous fluoroalcohol solutions and SDS micelles permitted their interactions with membrane-like environments to be explored. WaterLOGSY experiments and Mn2+-based paramagnetic probes were also applied to assess their orientations with respect to the SDS micelles. In addition, pulse-field gradient (PFG) diffusion NMR spectroscopy studies were conducted, under different experimental conditions (i.e., concentration, temperature) to characterize the possible changes in the peptides' aggregation states as a putative critical factor for their antimicrobial activity. Finally, molecular dynamics simulations on a variety of conformations showed the intrinsic flexibility of these peptides in both aqueous solutions and membrane-mimetic systemsThe authors wish to thank the Spanish Ministerio de Ciencia e Innovación (MICINN, SAF2008-01845), the Generalitat Valenciana (GVA, ACOMP/09/048), and the Centro de Investigación Príncipe Felipe for their economic supportPeer reviewe

MYC Induces a Hybrid Energetics Program Early in Cell Reprogramming

Summary: Cell reprogramming is thought to be associated with a full metabolic switch from an oxidative- to a glycolytic-based metabolism. However, neither the dynamics nor the factors controlling this metabolic switch are fully understood. By using cellular, biochemical, protein array, metabolomic, and respirometry analyses, we found that c-MYC establishes a robust bivalent energetics program early in cell reprogramming. Cells prone to undergo reprogramming exhibit high mitochondrial membrane potential and display a hybrid metabolism. We conclude that MYC proteins orchestrate a rewiring of somatic cell metabolism early in cell reprogramming, whereby somatic cells acquire the phenotypic plasticity necessary for their transition to pluripotency in response to either intrinsic or external cues. : Torres and colleagues describe an MYC-dependent remodeling of mitochondrial dynamics and metabolism during the first stage of cell reprogramming. Endogenous MYC activity was found to be necessary for cell reprogramming, likely by establishing a hybrid metabolic state characterized by elevated glycolytic flux and a somatic oxidative metabolic rate. MYC polarized mitochondria by increasing ATPIF1 and labeled cells prone to cell reprogramming. Keywords: cell reprogramming, metabolism, mitochondrial dynamics, cell signalin