Folding Kinetics and Volume Variation of the ß-Hairpin Peptide Chignolin upon Ultrafast pH-Jumps.

In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex biomacromolecules. We have previously reported on the folding/unfolding kinetics of a model α-helix. Here, we study folding transitions in chignolin (GYDPETGTWG), a short ß-hairpin peptide previously used as a model to study conformational changes in ß-sheet proteins. Although previously suggested, until now, the role of the Tyr2-Trp9 interaction in the folding mechanism of chignolin was not clear. In the present work, pH-dependent conformational changes of chignolin were characterized by circular dichroism (CD), nuclear magnetic resonance (NMR), ultrafast pH-jump coupled with time-resolved photoacoustic calorimetry (TR-PAC), and molecular dynamics (MD) simulations. Taken together, our results present a comprehensive view of chignolin's folding kinetics upon local pH changes and the role of the Tyr2-Trp9 interaction in the folding process. CD data show that chignolin's ß-hairpin formation displays a pH-dependent skew bell-shaped curve, with a maximum close to pH 6, and a large decrease in ß-sheet content at alkaline pH. The ß-hairpin structure is mainly stabilized by aromatic interactions between Tyr2 and Trp9 and CH-π interactions between Tyr2 and Pro4. Unfolding of chignolin at high pH demonstrates that protonation of Tyr2 is essential for the stability of the ß-hairpin. Refolding studies were triggered by laser-induced pH-jumps and detected by TR-PAC. The refolding of chignolin from high pH, mainly due to the protonation of Tyr2, is characterized by a volume expansion (10.4 mL mol-1), independent of peptide concentration, in the microsecond time range (lifetime of 1.15 µs). At high pH, the presence of the deprotonated hydroxyl (tyrosinate) hinders the formation of the aromatic interaction between Tyr2 and Trp9 resulting in a more disorganized and dynamic tridimensional structure of the peptide. This was also confirmed by comparing MD simulations of chignolin under conditions mimicking neutral and high pH.

An Intracellular Metabolic Signature as a Potential Donor-Independent Marker of the Osteogenic Differentiation of Adipose Tissue Mesenchymal Stem Cells.

This paper describes an untargeted NMR metabolomics study to identify potential intracellular donor-dependent and donor-independent metabolic markers of proliferation and osteogenic differentiation of human adipose mesenchymal stem cells (hAMSCs). The hAMSCs of two donors with distinct proliferating/osteogenic characteristics were fully characterized regarding their polar endometabolome during proliferation and osteogenesis. An 18-metabolites signature (including changes in alanine, aspartate, proline, tyrosine, ATP, and ADP, among others) was suggested to be potentially descriptive of cell proliferation, independently of the donor. In addition, a set of 11 metabolites was proposed to compose a possible donor-independent signature of osteogenesis, mostly involving changes in taurine, glutathione, methylguanidine, adenosine, inosine, uridine, and creatine/phosphocreatine, choline/phosphocholine and ethanolamine/phosphocholine ratios. The proposed signatures were validated for a third donor, although they require further validation in a larger donor cohort. We believe that this proof of concept paves the way to exploit metabolic markers to monitor (and potentially predict) cell proliferation and the osteogenic ability of different donors.

Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing Osteogenic Differentiation.

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.

NMR Metabolomics Assessment of Osteogenic Differentiation of Adipose-Tissue-Derived Mesenchymal Stem Cells.

This Article presents, for the first time to our knowledge, an untargeted nuclear magnetic resonance (NMR) metabolomic characterization of the polar intracellular metabolic adaptations of human adipose-derived mesenchymal stem cells during osteogenic differentiation. The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts, and untargeted metabolomics may unveil novel metabolic information on the osteogenic differentiation of MSCs, allowing their behavior to be understood and monitored/guided toward effective therapies. Our results unveiled statistically relevant changes in the levels of just over 30 identified metabolites, illustrating a highly dynamic process with significant variations throughout the whole 21-day period of osteogenic differentiation, mainly involving amino acid metabolism and protein synthesis; energy metabolism and the roles of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation; cell membrane metabolism; nucleotide metabolism (including the specific involvement of O-glycosylation intermediates and NAD+); and metabolic players in protective antioxidative mechanisms (such as glutathione and specific amino acids). Different metabolic stages are proposed and are supported by putative biochemical explanations for the metabolite changes observed. This work lays the groundwork for the use of untargeted NMR metabolomics to find potential metabolic markers of osteogenic differentiation efficacy.

Metabolomic Applications in Stem Cell Research: a Review.

This review describes the use of metabolomics to study stem cell (SC) characteristics and function, excluding SCs in cancer research, suited to a fully dedicated text. The interest in employing metabolomics in SC research has consistently grown and emphasis is, here, given to developments reported in the past five years. This text informs on the existing methodologies and their complementarity regarding the information provided, comprising untargeted/targeted approaches, which couple mass spectrometry or nuclear magnetic resonance spectroscopy with multivariate analysis (and, in some cases, pathway analysis and integration with other omics), and more specific analytical approaches, namely isotope tracing to highlight particular metabolic pathways, or in tandem microscopic strategies to pinpoint characteristics within a single cell. The bulk of this review covers the existing applications in various aspects of mesenchymal SC behavior, followed by pluripotent and neural SCs, with a few reports addressing other SC types. Some of the central ideas investigated comprise the metabolic/biological impacts of different tissue/donor sources and differentiation conditions, including the importance of considering 3D culture environments, mechanical cues and/or media enrichment to guide differentiation into specific lineages. Metabolomic analysis has considered cell endometabolomes and exometabolomes (fingerprinting and footprinting, respectively), having measured both lipid species and polar metabolites involved in a variety of metabolic pathways. This review clearly demonstrates the current enticing promise of metabolomics in significantly contributing towards a deeper knowledge on SC behavior, and the discovery of new biomarkers of SC function with potential translation to in vivo clinical practice.

Using amyloid autofluorescence as a biomarker for lysozyme aggregation inhibition.

The assembly of proteins into amyloidogenic aggregates underlies the onset and symptoms of several pathologies, including Alzheimer's disease, Parkinson's disease and type II diabetes. Among the efforts for fighting these diseases, there is a great demand for developing novel, fast and reliable methods for in vitro screening of new drugs that may suppress or reverse amyloidogenesis. Recent studies unravelled a progressive increase in a blue autofluorescence upon amyloid formation originated from many different proteins, including the peptide amyloid-ß, lysozyme or insulin. Herein, we propose a drug screening method using this property, avoiding the use of external probe dyes. We demonstrate that the inhibition of lysozyme amyloid formation by means of two known inhibitors, tartrazine and amaranth, can be monitored based on the autofluorescence of lysozyme amyloid aggregates. Our results show that amyloid luminescence is an intrinsic property that can be potentially applied in a screening assay, allowing the ranking of drug efficiency. The assays demonstrated here are fast to perform and suitable for scaling using microplate assays, configuring a new sensitive and economically feasible method.

One Peptide Reveals the Two Faces of α-Helix Unfolding-Folding Dynamics.

The understanding of fast folding dynamics of single α-helices comes mostly from studies on rationally designed peptides displaying sequences with high helical propensity. The folding/unfolding dynamics and energetics of α-helix conformations in naturally occurring peptides remains largely unexplored. Here we report the study of a protein fragment analogue of the C-peptide from bovine pancreatic ribonuclease-A, RN80, a 13-amino acid residue peptide that adopts a highly populated helical conformation in aqueous solution. 1H NMR and CD structural studies of RN80 showed that α-helix formation displays a pH-dependent bell-shaped curve, with a maximum near pH 5, and a large decrease in helical content in alkaline pH. The main forces stabilizing this short α-helix were identified as a salt bridge formed between Glu-2 and Arg-10 and the cation-π interaction involving Tyr-8 and His-12. Thus, deprotonation of Glu-2 or protonation of His-12 are essential for the RN80 α-helix stability. In the present study, RN80 folding and unfolding were triggered by laser-induced pH jumps and detected by time-resolved photoacoustic calorimetry (PAC). The photoacid proton release, amino acid residue protonation, and unfolding/folding events occur at different time scales and were clearly distinguished using time-resolved PAC. The partial unfolding of the RN80 α-helix, due to protonation of Glu-2 and consequent breaking of the stabilizing salt bridge between Glu-2 and Arg-10, is characterized by a concentration-independent volume expansion in the sub-microsecond time range (0.8 mL mol-1, 369 ns). This small volume expansion reports the cost of peptide backbone rehydration upon disruption of a solvent-exposed salt bridge, as well as backbone intrinsic expansion. On the other hand, RN80 α-helix folding triggered by His-12 protonation and subsequent formation of a cation-π interaction leads to a microsecond volume contraction (-6.0 mL mol-1, ∼1.7 µs). The essential role of two discrete side chain interactions, a salt bridge, and in particular a single cation-π interaction in the folding dynamics of a naturally occurring α-helix peptide is uniquely revealed by these data.

In situ ultra-fast heat deposition does not perturb the structure of serum albumin.

MnTPPS is a metallic water soluble porphyrin with high potential to be used as a contrast agent in photoacoustic tomography. In order to fully understand the interaction between MnTPPS and serum albumin and to investigate the effect of the light induced fast in situ heat deposition by MnTPPS in the protein, we performed several experimental studies using fluorescence and circular dichroism spectroscopies, as well as photoacoustic calorimetry. To identify the possible binding site(s) of the metalloporphyrin in serum albumin and to help interpret the spectroscopic results, a molecular docking exercise was also carried out. The fluorescence data indicate a 1 : 1 stoichiometry for the complex BSA : MnTPPS. The molecular docking results suggest one binding site at the subdomain IB of albumin, where Trp-134 is found, as the main binding site for MnTPPS. The CD data indicate no significant conformational changes of the BSA secondary structure upon MnTPPS binding and even after several minutes of laser excitation of MnTPPS. TR-PAC results show that the in situ heat deposition from MnTPPS does not cause any significant transient conformational change to the BSA structure. In conclusion, this work demonstrates that MnTPPS, in addition to the necessary physical and chemical properties to be used as a contrast agent in photoacoustic tomography, can be effectively carried by albumin and that in situ heat release following light absorption does not cause any significant damage to the protein structure.

A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation.

Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer's and Parkinson's. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo.

Evaluation by fluorescence, STD-NMR, docking and semi-empirical calculations of the o-NBA photo-acid interaction with BSA.

Serum albumins present reversible pH dependent conformational transitions. A sudden laser induced pH-jump is a methodology that can provide new insights on localized protein (un)folding processes that occur within the nanosecond to microsecond time scale. To generate the fast pH jump needed to fast-trigger a protein conformational event, a photo-triggered acid generator as o-nitrobenzaldehyde (o-NBA) can be conveniently used. In order to detect potential specific or nonspecific interactions between o-NBA and BSA, we have performed ligand-binding studies using fluorescence spectroscopy, saturation transfer difference (STD) NMR, molecular docking and semi-empirical calculations. Fluorescence quenching indicates the formation of a non-fluorescent complex in the ground-state between the fluorophore and the quencher, but o-NBA does not bind much effectively to the protein (Ka~4.34×10(3)M(-1)) and thus can be considered a relatively weak binder. The corresponding thermodynamic parameters: ΔG°, ΔS° and ΔH° showed that the binding process is spontaneous and entropy driven. Results of (1)H STD-NMR confirm that the photo-acid and BSA interact, and the relative intensities of the signals in the STD spectra show that all o-NBA protons are equally involved in the binding process, which should correspond to a nonspecific interaction. Molecular docking and semi-empirical calculations suggest that the o-NBA binds preferentially to the Trp-212-containing site of BSA (FA7), interacting via hydrogen bonds with Arg-217 and Tyr-149 residues.

A novel bis-furan scaffold for transthyretin stabilization and amyloid inhibition.

The design and synthesis of a novel bis-furan scaffold tailored for high efficiency at inhibiting transthyretin amyloid formation is reported. In vitro results show that the discovered compounds are more efficient inhibitors of amyloid formation than tafamidis, a drug currently used in the treatment of familial amyloid polyneuropathy (FAP), despite their lower molecular weight and lipophilicity. Moreover, ex vivo experiments with the strongest inhibitor in the series, conducted in human blood plasma from normal and FAP Val30Met-transthyretin carriers, disclose remarkable affinity and selectivity profiles. The promises and challenges facing further development of this compound are discussed under the light of increasing evidence implicating transthyretin stability as a key factor not only in transthyretin amyloidoses and several associated co-morbidities, but also in Alzheimer's disease.

A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model.

The aggregation of proteins into insoluble amyloid fibrils is the hallmark of many, highly debilitating, human pathologies such as Alzheimer's or Parkinson's disease. Transthyretin (TTR) is a homotetrameric protein implicated in several amyloidoses like Senile Systemic Amyloidosis (SSA), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), and the rare Central Nervous System selective Amyloidosis (CNSA). In this work, we have investigated the kinetics of TTR aggregation into amyloid fibrils produced by the addition of NaCl to acid-unfolded TTR monomers and we propose a mathematically simple kinetic mechanism to analyse the aggregation kinetics of TTR. We have conducted circular dichroism, intrinsic tryptophan fluorescence and thioflavin-T emission experiments to follow the conformational changes accompanying amyloid formation at different TTR concentrations. Kinetic traces were adjusted to a two-step model with the first step being second-order and the second being unimolecular. The molecular species present in the pathway of TTR oligomerization were characterized by size exclusion chromatography coupled to multi-angle light scattering and by transmission electron microscopy. The results show the transient accumulation of oligomers composed of 6 to 10 monomers in agreement with reports suggesting that these oligomers may be the causative agent of cell toxicity. The results obtained may prove to be useful in understanding the mode of action of different compounds in preventing fibril formation and, therefore, in designing new drugs against TTR amyloidosis.

VEGF-functionalized dextran has longer intracellular bioactivity than VEGF in endothelial cells.

Herein, we report that VEGF-functionalized dextran (dexOx-VEGF) is comparatively superior to free VEGF in prolonging the phosphorylation of VEGF receptor 2 (VEGFR-2). Both dexOx-VEGF and free VEGF activate VEGFR-2, and the complexes are internalized into early endosomes (EEA1(+) vesicles) and then transported to lysosomes (Rab7(+) vesicles). However, after cell activation, dexOx-VEGF is preferentially colocalized in early endosomes where VEGF signaling is still active while free VEGF is preferentially transported to late endosomes or lysosomes. We further show that dexOx-VEGF after phosphorylation of VEGF receptor 2 induces an increase of intracellular Ca(2+) and activates VEGF downstream effectors such as Akt and extracellular signal-regulated kinase (ERK1/2) proteins. Under specific conditions, the activation level is different from the one observed for free VEGF, thus suggesting mechanistic differences, which is illustrated by cell migration and cord-like formation studies. DexOx-VEGF can be cross-linked with adipic acid dihydrazide to form a degradable gel, which in turn can be incorporated in a fibrin gel containing endothelial cells (ECs) to modulate their activity. We envision that these constructs might be beneficial to extend the pro-angiogenic activity of VEGF in ischemic tissues and to modulate the biological activity of vascular cells.

Changes in channel trafficking and protein stability caused by LQT2 mutations in the PAS domain of the HERG channel.

Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay.