Folding of the nascent polypeptide chain of a histidine phosphocarrier protein in vitro

The phosphotransferase system (PTS), a metabolic pathway formed by five proteins, modulates the use of sugars in bacteria. The second protein in the chain is the histidine phosphocarrier, HPr, with the binding site at His15. The HPr kinase/phosphorylase (HPrK/P), involved in the bacterial use of carbon sources, phosphorylates HPr at Ser46, and it binds at its binding site. The regulator of sigma D protein (Rsd) also binds to HPr at His15. We have designed fragments of HPr, growing from its N-terminus and containing the His15. In this work, we obtained three fragments, HPr38, HPr58 and HPr70, comprising the first thirty-eight, fifty-eight and seventy residues of HPr, respectively. All fragments were mainly disordered, with evidence of a weak native-like, helical population around the binding site, as shown by fluorescence, far-ultraviolet circular dichroism, size exclusion chromatography and nuclear magnetic resonance. Although HPr38, HPr58 and HPr70 were disordered, they could bind to: (i) the N-terminal domain of first protein of the PTS, EIN; (ii) Rsd; and, (iii) HPrK/P, as shown by fluorescence and biolayer interferometry (BLI). The association constants for each protein to any of the fragments were in the low micromolar range, within the same range than those measured in the binding of HPr to each protein. Then, although acquisition of stable, native-like secondary and tertiary structures occurred at the last residues of the polypeptide, the ability to bind protein partners happened much earlier in the growing chain. Binding was related to the presence of the native-like structure around His15

The muscle-relaxing C-terminal peptide from troponin I populates a nascent helix, facilitating binding to tropomyosin with a potent therapeutic effect

The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184-210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca2+-sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnIC27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (aTm), with a detectably higher affinity (~10 µM) of HcTnIC27 than that of HcTnI-C27-H (~15 µM), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to aTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca2+-desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart

Mild Muscle Mitochondrial Fusion Distress Extends Drosophila Lifespan through an Early and Systemic Metabolome Reorganization

[EN] In a global aging population, it is important to understand the factors affecting systemic aging and lifespan. Mitohormesis, an adaptive response caused by different insults affecting the mitochondrial network, triggers a response from the nuclear genome inducing several pathways that promote longevity and metabolic health. Understanding the role of mitochondrial function during the aging process could help biomarker identification and the development of novel strategies for healthy aging. Herein, we interfered the muscle expression of the Drosophila genes Marf and Opa1, two genes that encode for proteins promoting mitochondrial fusion, orthologues of human MFN2 and OPA1. Silencing of Marf and Opa1 in muscle increases lifespan, improves locomotor capacities in the long term, and maintains muscular integrity. A metabolomic analysis revealed that muscle down-regulation of Marf and Opa1 promotes a non-autonomous systemic metabolome reorganization, mainly affecting metabolites involved in the energetic homeostasis: carbohydrates, lipids and aminoacids. Interestingly, the differences are consistently more evident in younger flies, implying that there may exist an anticipative adaptation mediating the protective changes at the older age. We demonstrate that mild mitochondrial muscle disturbance plays an important role in Drosophila fitness and reveals metabolic connections between tissues. This study opens new avenues to explore the link of mitochondrial dynamics and inter-organ communication, as well as their relationship with muscle-related pathologies, or in which muscle aging is a risk factor for their appearance. Our results suggest that early intervention in muscle may prevent sarcopenia and promote healthy aging.Work in the laboratory of M.I.G. was funded by PROMETEU/2018/135 from "Conselleria, de Sanitat de la Generalitat Valenciana". Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020).Tapia, A.; Palomino-Schätzlein, M.; Roca, M.; Lahoz, A.; Pineda-Lucena, A.; López Del Amo, V.; Galindo, MI. (2021). Mild Muscle Mitochondrial Fusion Distress Extends Drosophila Lifespan through an Early and Systemic Metabolome Reorganization. International Journal of Molecular Sciences. 22(22):1-20. https://doi.org/10.3390/ijms222212133120222

Metabolic footprint of aging and obesity in red blood cells

Aging; Metabolomics; ObesityEnvelliment; Metabolòmica; ObesitatEnvejecimiento; Metabolómica; ObesidadAging is a physiological process whose underlying mechanisms are still largely unknown. The study of the biochemical transformations associated with aging is crucial for understanding this process and could translate into an improvement of the quality of life of the aging population. Red blood cells (RBCs) are the most abundant cells in humans and are involved in essential functions that could undergo different alterations with age. The present study analyzed the metabolic alterations experienced by RBCs during aging, as well as the influence of obesity and gender in this process. To this end, the metabolic profile of 83 samples from healthy and obese patients was obtained by Nuclear Magnetic Resonance spectroscopy. Multivariate statistical analysis revealed differences between Age-1 (≤45) and Age-2 (>45) subgroups, as well as between BMI-1 (<30) and BMI-2 (≥30) subgroups, while no differences were associated with gender. A general decrease in the levels of amino acids was detected with age, in addition to metabolic alterations of glycolysis, the pentose phosphate pathway, nucleotide metabolism, glutathione metabolism and the Luebering-Rapoport shunt. Obesity also had an impact on the metabolomics profile of RBCs; sometimes mimicking the alterations induced by aging, while, in other cases, its influence was the opposite, suggesting these changes could counteract the adaptation of the organism to senescence.This work was supported by the Carlos III Health Institute and the European Regional Development Fund (PI16/02064 and PI20/01588), the Agency for Management of University and Research Grants (AGAUR) of Catalonia (2017SGR1303) and the Ministry of Economy and Competitiveness (SAF2017-89229-R). Equipment employed in this work was partially funded by Generalitat Valenciana and ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020)

Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome

From MDPI via Jisc Publications RouterHistory: accepted 2021-11-17, pub-electronic 2021-11-18Publication status: PublishedFunder: Generalitat Valenciana; Grant(s): PROMETEU/2018/135Funder: European Commission; Grant(s): ERDF of Comunitat Valenciana 2014–2020Funder: ApoyoDravet; Grant(s): N/ADravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments

Identification of Bioactive Compounds in Polar and Nonpolar Extracts of Araujia sericifera

Araujia sericifera is a native perennial, climbing laticiferous shrub from South America that is currently naturalized in many other countries. Previous data describe promising properties for A. sericifera, but no systematic study of its bioactive compounds and possible medicinal applications has been conducted to date. In the present study, aerial parts of A. sericifera (leaves, stems, and fruits) were explored by combining GC-MS and NMR spectroscopy analysis for both nonpolar (hexane) and polar (methanol) extracts. The hexanic extracts contained high amounts of pentacyclic triterpenes including two new metabolites, 3-tigloyl germanicol (18) and 3-tigloyl lupeol (19). The methanolic extracts revealed the presence of luteolin-7-glucoside (24), trigonelline (22), and conduritol F (23) as the main constituents. A multivariate study of a meaningful number of extracts allowed us to determine the distribution of compounds inside the plant. A cytotoxic evaluation in vitro showed that both leaf and fruit hexanic extracts presented a moderate activity against human breast carcinoma cell lines (MDA-MB-453 and MCF-7) and human colon carcinoma cell line (HCT-116) by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay

Metabolic fingerprint of acromegaly and its potential usefulness in clinical practice

Acromegaly; Metabolomics; Amino acidsAcromegalia; Metabolómica; AminoácidosAcromegàlia; Metabolòmica; AminoàcidsInsulin-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.This research was funded by Pfizer Global Investigator Initiated Research. J.P.C. and R.M.L. are funded by Junta de Andalucía (CTS-1406, BIO-0139), Ministerio de Ciencia, Innovación y Universidades (BFU2016-80360-R), and Instituto de Salud Carlos III, co-funded by European Union (ERDF/ESF, “Investing in your future”: PI16/00264, CP15/00156 and CIBERobn). CIBER is an initiative of Instituto de Salud Carlos III

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI 2018-097991-B-I00 to JN and PGC 2018-094548-B-I00 to AA and PM), and the Basque Government (IT1175-19 to AA and PM)

A Drosophila model of GDAP1 function reveals the involvement of insulin signalling in the mitochondria-dependent neuromuscular degeneration

[EN] Charcot-Marie-Tooth disease is a rare peripheral neuropathy for which there is no specific treatment. Some forms of Charcot-Marie-Tooth are due to mutations in the GDAP1 gene. A striking feature of mutations in GDAP1 is that they have a variable clinical manifestation, according to disease onset and progression, histology and mode of inheritance. Studies in cellular and animal models have revealed a role of GDAP1 in mitochondrial morphology and distribution, calcium homeostasis and oxidative stress. To get a better understanding of the disease mechanism we have generated models of over-expression and RNA interference of the Drosophila Gdapl gene. In order to get an overview about the changes that Gdapl mutations cause in our disease model, we have combined a comprehensive determination of the metabolic profile in the flies by nuclear magnetic resonance spectroscopy with gene expression analyses and biophysical tests. Our results revealed that both up- and down-regulation of Gdapl results in an early systemic inactivation of the insulin pathway before the onset of neuromuscular degeneration, followed by an accumulation of carbohydrates and an increase in the (3-oxidation of lipids. Our findings are in line with emerging reports of energy metabolism impairments linked to different types of neural pathologies caused by defective mitochondrial function, which is not surprising given the central role of mitochondria in the control of energy metabolism. The relationship of mitochondrial dynamics with metabolism during neurodegeneration opens new avenues to understand the cause of the disease, and for the discovery of new biomarkers and treatments.This work was supported by a project grant from the Association Francaise contre les Myopathies [AFM 18540 to M.I.G]; a collaborative grant from International Rare Diseases Research consortium (IRDiRC) and Institute de Salud Carlos III [IR11/TREAT-CMT to M.I.G. (partner 12) and F.V.P. (partner 8)]; funding from Institute de Salud Carlos III through Biomedical Network Research Center for Rare Diseases and the INGENIO 2010 program to F.V.P.; and a project grant from the Spanish Government (Secretaria de Estado de Investigacion, Desarollo e Innovacion, Ministerio de Economia y Competitividad) [SAF2014-53977-R to A.P.].Lopez Del Amo, V.; Palomino-Schätzlein, M.; Seco-Cervera, M.; Garcia-Gimenez, JL.; Pallardó-Calatayud, FV.; Pineda-Lucena, A.; Galindo-Orozco, MI. (2017). A Drosophila model of GDAP1 function reveals the involvement of insulin signalling in the mitochondria-dependent neuromuscular degeneration. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1863(3):801-809. https://doi.org/10.1016/j.bbadis.2017.01.003S8018091863