Integrative miRNA and whole-genome analyses of epicardial adipose tissue in patients with coronary atherosclerosis.

BACKGROUND: Epicardial adipose tissue (EAT) is an atypical fat depot surrounding the heart with a putative role in the development of atherosclerosis. METHODS AND RESULTS: We profiled genes and miRNAs in perivascular EAT and subcutaneous adipose tissue (SAT) of metabolically healthy patients without coronary artery disease (CAD) vs. metabolic patients with CAD. Compared with SAT, a specific tuning of miRNAs and genes points to EAT as a tissue characterized by a metabolically active and pro-inflammatory profile. Then, we depicted both miRNA and gene signatures of EAT in CAD, featuring a down-regulation of genes involved in lipid metabolism, mitochondrial function, nuclear receptor transcriptional activity, and an up-regulation of those involved in antigen presentation, chemokine signalling, and inflammation. Finally, we identified miR-103-3p as candidate modulator of CCL13 in EAT, and a potential biomarker role for the chemokine CCL13 in CAD. CONCLUSION: EAT in CAD is characterized by changes in the regulation of metabolism and inflammation with miR-103-3p/CCL13 pair as novel putative actors in EAT function and CAD.

Prohibitin: A Novel Molecular Player in KDEL Receptor Signalling.

The KDEL receptor (KDELR) is a seven-transmembrane-domain protein involved in retrograde transport of protein chaperones from the Golgi complex to the endoplasmic reticulum. Our recent findings have shown that the Golgi-localised KDELR acts as a functional G-protein-coupled receptor by binding to and activating Gs and Gq. These G proteins induce activation of PKA and Src and regulate retrograde and anterograde Golgi trafficking. Here we used an integrated coimmunoprecipitation and mass spectrometry approach to identify prohibitin-1 (PHB) as a KDELR interactor. PHB is a multifunctional protein that is involved in signal transduction, cell-cycle control, and stabilisation of mitochondrial proteins. We provide evidence that depletion of PHB induces intense membrane-trafficking activity at the ER-Golgi interface, as revealed by formation of GM130-positive Golgi tubules, and recruitment of p115, ß-COP, and GBF1 to the Golgi complex. There is also massive recruitment of SEC31 to endoplasmic-reticulum exit sites. Furthermore, absence of PHB decreases the levels of the Golgi-localised KDELR, thus preventing KDELR-dependent activation of Golgi-Src and inhibiting Golgi-to-plasma-membrane transport of VSVG. We propose a model whereby in analogy to previous findings (e.g., the RAS-RAF signalling pathway), PHB can act as a signalling scaffold protein to assist in KDELR-dependent Src activation.

Functional amyloids in insect immune response.

The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention.

Proteomics for the discovery of nuclear bile acid receptor FXR targets.

Nuclear receptors (NRs) are important pharmacological targets for a number of diseases, including cancer and metabolic disorders. To unmask the direct role of NR function it is fundamental to find the NR targets. During the last few years several NRs have been shown to affect microRNA expression, thereby modulating protein levels. The farnesoid X receptor (FXR), the main regulator of bile acid (BA) homeostasis, also regulates cholesterol, lipid and glucose metabolism. Here we used, for the first time, a proteomics approach on mice treated with a FXR ligand to find novel hepatic FXR targets. Nineteen spots with a more than two-fold difference in protein amounts were found by 2D-DIGE and 20 proteins were identified by MALDI-TOF MS as putative novel FXR targets. The most striking feature of the protein list was the great number of mitochondrial proteins, indicating a substantial impact of FXR activation on mitochondrial function in the liver. To examine if the differences found in the proteomics assay reflected differences at the mRNA level, a microarray assay was generated on hepatic samples from wild type and FXR(-/-) mice treated with a FXR ligand and compared to vehicle treatment. At least six proteins were shown to be regulated only at a post-transcriptional level. In conclusion, our study provides the impetus to include proteomic analysis for the identification of novel targets of transcription factors, such as NRs. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Investigating by circular dichroism some amyloidogenic elastin-derived polypeptides.

Tamburro and coworkers have demonstrated that some elastin-derived polypeptide sequences are able to give rise, in vitro, to amyloid-like fibers. The biological relevance of this finding could be explained by the recent detection of some amyloidogenic material found in arteries of old patients affected by atherosclerosis and demonstrated to be elastin derived. In this context, the comprehension of the mechanism responsible for the amyloid-like fibrillogenesis of elastin-derived sequences is of crucial importance for the design of drugs that could inhibit the amyloidogenic process. To gain further insights into the elastin amyloidogenic process, we studied the polypeptide sequences encoded by Exon 7 and Exon 32 of the human tropoelastin gene, and we demonstrated that only Exon 32 is able to aggregate in amyloid-like fibers. Vis-UV Thioflavin T circular dichroism (CD) spectroscopy rapidly and unambiguously detected the amyloidogenic propensity of the polypeptides. To gain additional insights into the aggregation mechanism of elastin-derived amyloidogenic peptides, we carried out the kinetics of EX32 amyloid-like aggregates by using ThT dye. CD spectroscopy was also used for investigating the secondary structure of the polypeptides, thus giving useful insights into the conformations involved in amyloid-like fiber formation. Furthermore, complementary techniques such as fluorescence spectroscopy, spectral shift, and binding Congo red UV assays as well as atomic force microscopy were also used to confirm the amyloidogenic behavior of the studied polypeptides.

Molecular and supramolecular structural studies on significant repetitive sequences of resilin.

Resilin is a member of the family of elastomeric proteins and is found in specialised regions of the cuticle of most insects, and provides low stiffness, high strain and efficient energy storage. It is best known for its role in insect flight and the remarkable jumping ability of fleas and spittle bugs. In common with other elastomeric proteins, the recently identified Drosophila melanogaster proresilin shows glycine-rich repetitive sequences; in particular the N- and C-terminal regions of the protein are dominated by 18 repeats of a 15-residue sequence (SDTYGAPGGGNGGRP) and eleven repeats of a 13-residue sequence (GYSGGRPGGQDLG), respectively. We synthesised and analysed the molecular and supramolecular structure of some polypeptides with sequences belonging to the glycine-rich repeated domain of D. melanogaster resilin. The conformational studies performed by CD, FTIR and NMR spectroscopies pointed to the coexistence of two main conformational features, such as folded beta-turns and (quasi)extended structures (e.g., poly-L-proline II conformation) in common with other elastomeric proteins; this suggests an elasticity mechanism for resilin common to other elastomeric proteins. Our data show that also in the case of resilin, repetitive sequences are characterised by autonomous structures almost independent of the remaining parts of the molecule as already extensively found for elastin. From a supramolecular point of view, a great tendency to aggregate in fibrous structures is observed, particularly for the resilin- inspired polypeptide (PGGGN)(10). This is encouraging for the development of resilin-based biomaterials for the production of biocompatible medical devices, as well as high performing elastic materials.

Human tropoelastin sequence: dynamics of polypeptide coded by exon 6 in solution.

Calorimetric studies were performed on exon 6 in powdered form and in solution [water and 2,2,2-trifluoroethanol (TFE), a structure-inducing solvent or cosolvent]. Dynamic dielectric spectroscopy (DDS) analyses were realized in water and 20% TFE. The major role of solvent-peptide organization is evidenced with these techniques. Calorimetric measurements reveal the structural water organization around the polypeptide as well as the presence of hydrophobic interactions in TFE solution. Dielectric measurements showed for exon 6/water a decrease of relaxations times of bulk solvent implying a faster dynamics with a slight increase of the activation entropy, suggesting that exon 6 probably creates disorder within the solvent. For TFE/water mixtures, an influence of exon 6 on its environment was seen with a relaxation associated with the exon 6/solvent interactions reinforced by storage of 72 h. Finally, exon 6/solvent interactions were clearly observed with addition of TFE.

A never-ending love story with elastin: a scientific autobiography.

The author describes, in a quite unconventional way, the most important results achieved in the last 50 years in the field of elastin structure-elasticity relationships, beginning with the first invaluable findings of Partridge on desmosines and isodesmosines until the most recent theories on elastomeric proteins. The author also relates a scientific autobiography characterized by his greatest passion, elastin.

Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome.

Mono-ADP-ribosylation is a reversible posttranslational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. For the latter, both the enzymes and their targets have largely remained elusive, mainly due to the lack of specific techniques to study this reaction. The recent discovery of the macro domain, a protein module that interacts selectively with ADP-ribose, prompted us to investigate whether this interaction can be extended to the identification of ADP-ribosylated proteins. Here, we report that macro domains can indeed be used as selective baits for high-affinity purification of mono-ADP-ribosylated proteins, which can then be identified by mass spectrometry. Using this approach, we have identified a series of cellular targets of ADP-ribosylation reactions catalyzed by cellular ADP-ribosyltransferases and toxins. These proteins include most of the known targets of ADP-ribosylation, indicating the validity of this method, and a large number of other proteins, which now need to be individually validated. This represents an important step toward the discovery of new ADP-ribosyltransferase targets and an understanding of the physiological role and the pharmacological potential of this protein modification.

On enhancers and inhibitors of elastin-derived amyloidogenesis.

AIMS: The main aim of this study is to better understand the self-aggregation mechanism of amyloid-like elastin-derived fibers in order to design and produce new powerful drugs that will inhibit the onset of 'amyloidosis'. MATERIALS & METHODS: Atomic force microscopy (AFM), Congo Red birefringence assay and Thioflavin T fluorescence measurements were used to demonstrate the amyloid-like behavior of some fragments of elastin protein (exon 30 [EX30] and exon 28 [EX28]). Turbidimetry on apparent absorbance technique was used to investigate the effect either of enhancers or of inhibitors on the amyloidogenic elastin-like peptides. Circular-dichroism spectroscopy was used to study the secondary structures of the peptides. RESULTS & DISCUSSION: We used Congo Red birefringence assay, Thioflavin T fluorescence measurements and AFM measurements that are used commonly to demonstrate the formation of amyloids. The elastin fibrillogenesis is amyloid-like. Then, the elastin amyloidogenesis is inhibited by particular pentapeptides. CONCLUSIONS: We have reported herein that the fibrillogenesis of elastin-derived EX28 and EX30 polypeptides is facilitated significantly by the effect of sodium taurocholate bile salt and is inhibited by a classical inhibitor of Abeta-amyloid peptide, such as KLVFF, as well as by novel inhibitors, designed by us on the basis of some elastin sequences.

Exon 26-coded polypeptide: an isolated hydrophobic domain of human tropoelastin able to self-assemble in vitro.

Hydrophobic domains of human tropoelastin are able to aggregate in a variegated manner. Some aggregates have typical features of the whole protein while others show peculiar self-assembling profiles. Among these hydrophobic domains, an important role in the self-assembling properties of tropoelastin in vitro could be assigned to the peptide encoded by exon 26 of the human tropoelastin gene, that, although unstructured in solution, has great tendency to self-assemble in an ordered manner. The present report describes the aggregation properties of this hydrophobic domain of human tropoelastin analysed by different ultra-structural approaches. Transmission electron microscopy shows that the peptide is able to form different aggregation entities from short rods to very long and flexible fibers, depending on the temperature and on the incubation time. At a microm scale, very long fibers as well as fractal aggregation patterns were observed. Data show that the isolated domain encoded by exon 26 of the tropoelastin gene is able to aggregate in a manner very similar to the whole tropoelastin protein. The aggregation properties are due to the peculiar sequence of EX26, and not to its amino acid composition, as evidenced by the supramolecular analysis of a scrambled sequence of exon 26-coded domain of human tropoelastin, showing a quite different aggregation patterns. These findings confirm that specific sequences can play a driving role in the aggregation process of tropoelastin molecule, at least in vitro, and indicate exon 26-encoded domain among these sequences.

Amyloid-like fibrils in elastin-related polypeptides: structural characterization and elastic properties.

We report on the structural characterization of amyloid-like fibrils, self-assembled from synthetic polypentapeptides poly(ValGlyGlyLeuGly), whose monomeric sequence is a recurring, simple building block of elastin. This polymer adopts a beta-sheet structure as revealed by circular dichroism and Fourier transform infrared spectroscopy. Furthermore, Thioflavin-T and Congo red birefringence assays confirm the presence of amyloid-like structures. To analyze the supramolecular assembly and elastic properties of the fibrils, we employed atomic force microsocopy and spectroscopy, measuring also the elasticity of mature elastin for a comparative analysis. In the case of fibrils we estimated a Young's modulus ranging from 3.5 to 7 MPa, whereas for elastin it is around 1 MPa. The possibility to section individual fibrils with nanometric control by the AFM tip, realizing biomolecular gaps in the 100 nm range, is also demonstrated. These results are expected to open interesting perspectives for the fabrication of protein-inspired nanostructures with specific physical and chemical properties for applications in biotechnology and tissue engineering.

Investigating by CD the molecular mechanism of elasticity of elastomeric proteins.

Elastomeric proteins are widespread in the animal kingdom, and their main function is to confer elasticity and resilience to organs and tissues. Besides common functional properties, elastomeric proteins share a common sequence design. They are usually constituted by repetitive sequences with a high content of glycine residues. From a conformational point of view, all the elastomeric proteins since now analyzed show a dynamic equilibria between folded (mainly beta-turns) and extended (polyproline II and beta-strands) conformations that could be at the origin of the high entropy of the relaxed state. As a matter of fact, elastin, lamprin, abductin, as well as the PEVK domain of titin share the same conformational ensemble, thus pointing to a common molecular mechanism as the origin of elasticity. CD spectroscopy represents the proper spectroscopic technique to be used overall because of its particular sensitivity to the presence of PPII structure. Its use in the molecular studies of elastin, abductin, and lamprin as well as the recently analyzed protein resilin will be presented.

Charge transport and intrinsic fluorescence in amyloid-like fibrils.

The self-assembly of polypeptides into stable, conductive, and intrinsically fluorescent biomolecular nanowires is reported. We have studied the morphology and electrical conduction of fibrils made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly). These amyloid-like nanofibrils, with a diameter ranging from 20 to 250 nm, result from self-assembly in aqueous solution at neutral pH. Their morphological properties and conductivity have been investigated by atomic force microscopy, scanning tunneling microscopy, and two-terminal transport experiments at the micro- and nanoscales. We demonstrate that the nanofibrils can sustain significant electrical conduction in the solid state at ambient conditions and have remarkable stability. We also show intrinsic blue-green fluorescence of the nanofibrils by confocal microscopy analyses. These results indicate that direct (label-free) excitation can be used to investigate the aggregation state or the polymorphism of amyloid-like fibrils (and possibly of other proteinaceous material) and open up interesting perspectives for the use of peptide-based nanowire structures, with tunable physical and chemical properties, for a wide range of nanobiotechnological and bioelectronic applications.

Investigating the amyloidogenic nanostructured sequences of elastin: sequence encoded by exon 28 of human tropoelastin gene.

In this paper we demonstrate that the sequence encoded by exon 28 (EX28) of human tropoelastin gene is able to give amyloid-like fibrils. CD (circular dichroism) in solution and solid-state FTIR (Fourier transform infrared spectroscopy) spectroscopies have shown the presence of beta-sheet conformation. At the supramolecular level the fibers formed by EX28 peptide were investigated by AFM (atomic force microscopy) and ESEM (environmental scanning electron microscopy). A very big left-handed helix, 100 mum long, is visible together with aggregates of different sizes, some of them being constituted by helically interwoven fibers. Furthermore, an additional AFM image of EX28 is shown where the ultrastructure found is somewhat reminiscent of a more or less retiform film. These findings should be useful for designing proper elastin-inspired biomaterials.

Supramolecular organization of elastin and elastin-related nanostructured biopolymers.

The ultrastructure of elastin has been extensively analyzed by different methodologies. Starting from the first descriptions, where elastin was depicted as an amorphous structure, more complex and, in some cases, varied morphologies were revealed. The supramolecular structures found for elastin have been compared with those found for other elastin-related polypeptides, such as alpha-elastin and tropoelastin, and very similar features emerged. This review will deal with the supramolecular organization exhibited by many elastin-related compounds, starting from elastin, going through polypeptides constituted by different domains of tropoelastin, up to polymers containing repetitive sequences of elastin. In particular, recent developments on biopolymers of general type poly(Val-Pro-Gly-Xaa-Gly) and poly(Xaa-Gly-Gly-Zaa-Gly) (Xaa, Zaa = Val, Leu, Lys, Glu, Orn) obtained either by chemical synthesis or recombinant DNA techniques will be discussed in detail. The general aim is to describe the supramolecular features useful for the identification of elastin-inspired nanostructured biopolymers for developing highly functional and biocompatible vascular grafts as well as scaffolds for tissue regeneration.

Molecular and supramolecular structural studies on human tropoelastin sequences.

One of the unusual properties of elastin is its ability to coacervate, which has been proposed to play an important role in the alignment of monomeric elastin for cross-linking into the polymeric elastin matrix. The temperature at which this transition takes place depends on several factors including protein concentration, ionic strength, and pH. Previously, polypeptide sequences encoded by different exons of the human tropoelastin gene have been analyzed for their ability to coacervate and to self-assemble. Few of them were indeed able to coacervate and only one, that encoded by exon 30 (EX30), gave amyloid fibers. In this article, we report on two chemically synthesized peptides-a decapeptide and an octadecapeptide-whose sequences are contained in the longer EX30 peptide and on a polypeptide (EX1-7) of 125 amino-acid residues corresponding to the sequence coded by the exons 1-7 and on a polypeptide (EX2-7) of 99 amino-acid residues encoded by exons 2-7 of human tropoelastin obtained by recombinant DNA techniques. Molecular and supramolecular structural characterization of these peptides showed that a minimum sequence of approximately 20 amino acids is needed to form amyloid fibers in the exon 30-derived peptides. The N-terminal region of mature tropoelastin (EX2-7) gives rise to a coacervate and forms elastinlike fibers, whereas the polypeptide sequence containing the signal peptide (EX1-7) forms mainly amyloid fibers. Circular dichroism spectra show that beta-structure is ubiquitous in all the sequences studied, suggesting that the presence of a beta-structure is a necessary, although not sufficient, requirement for the appearance of amyloid fibers.

Localizing alpha-helices in human tropoelastin: assembly of the elastin "puzzle".

Polyalanine cross-linking domains encoded by exons 6, 15, 17, 19, 21, 23, 25, 27, 29, 31 of human tropoelastin were synthesized, and their conformations were studied in different solutions and at different temperatures by CD and (1)H NMR. The results demonstrated the presence of poly-proline II helix (PPII) in aqueous solvent and of alpha-helical conformation in TFE. The (1)H NMR results allowed the precise localization of the helices along the peptide sequence. These data were further refined by prediction algorithms in order to take into account the reduced helix stability at the end of the peptides. Furthermore, the influence of flanking residues was checked by synthesizing and by determining the structure of a peptide spanning exon 31 coded domain and the first five residues of the following exon 32 coded domain. These studies, together with those previously published [Tamburro, A. M., Bochicchio, B., and Pepe, A. (2003) Biochemistry 42, 13147-62], are used to propose a coherent recomposition of the elastin pieces (domains) in order to give an acceptable solution to the elastin structure-function problem.

Kinetics and thermodynamics of type VIII beta-turn formation: a CD, NMR, and microsecond explicit molecular dynamics study of the GDNP tetrapeptide.

We report an experimental and theoretical study on type VIII beta-turn using a designed peptide of sequence GDNP. CD and NMR studies reveal that this peptide exists in equilibrium between type VIII beta-turn and extended conformations. Extensive MD simulations give a description of the free energy landscape of the peptide in which we retrieve the same two main conformations suggested by the experiments. The free energy difference between the two conformational states is very small and the transition between them occurs within a few kT at 300 K on a nanosecond timescale. The equilibrium is mainly driven by entropic contribution, which favors extended conformations over beta-turns. This confirms other theoretical studies showing that beta-turns are marginally stable in water solution because of the larger entropy of the extended state unless some stabilizing interactions exist. Our observations may be extended to any type of beta-turn and have important consequences for protein folding. A comparison of our MD and CD results also suggests a possible type VIII beta-turn CD signature indicated by one main band at 200 nm, close to that of random coil, and a fairly large shoulder at 220 nm. Last, our results clearly show that the XXXP motif can only fold into a type VIII beta-turn, which is consistent with its fairly strong propensity for this type of turn. This important finding may help for peptide design and is in line with recent studies on bioactive elastin peptides.

The dissection of human tropoelastin: from the molecular structure to the self-assembly to the elasticity mechanism.

After a historical introduction the authors describe their most recent results on the structure, assembly and elasticity of elastin. Recent results obtained by analyzing the conformation of polypeptide sequences encoded by the single exons of human tropoelastin demonstrated the presence of labile conformations such as poly-proline II helix (PPII) and beta-turns whose stability is strongly dependent on the microenvironment. Stable, periodic structures, such as alpha-helices, are only present in the poly-alanine cross-linking domains. These findings give a strong experimental basis to the understanding of the molecular mechanism of elasticity of elastin. In particular, they strongly support the description of the native relaxed state of the protein in terms of trans-conformational equilibria between extended and folded structures as previously proposed [Int. J. Biochem. Cell. Biol. 31 (1999) 261]. The same polypeptide sequences have been analyzed for their ability to coacervate and to self-assembly. Although the great majority of them were shown to be able to adopt more or less organized structures, only a few were indeed able to coacervate. Studies carried out by transmission electron microscopy showed the polypeptides to adopt a variety of supramolecular structures going from a filamentous organization (typical of elastin) to amyloid-like fibers. On the whole, the results obtained gave significant insight to the roles played by specific polypeptide sequences in self-assembly and possibly in elasticity.