Sequence and Structure Determinants for the Self-aggregation of Recombinant Polypeptides Modeled after Human Elastin

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.

Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo

Assembly and disassembly sequences were planned and assessed independently. However, as eco-friendly product design has been enforced by government regulation, it is needed to plan assembly and disassembly sequence at the same time in early product design stage. This paper aims to propose a sequence structure design method by integrating both assembly and disassembly sequence planning. First, graphs represent feasible assembly sequence with components. Second, adjacency matrices are developed to accumulate the sequence information for computational analysis. Third, a black box diagram is applied to understand and consider the functions of a product during assembly and disassembly operations. Finally, factors are determined to overcome gaps between each assembly and disassembly sequence structure. By considering product architecture with Design Structure Matrix (DSM), we can determine modular design for selective disassembly and interface type for assembly. A case study is performed with two coffee makers to demonstrate the effectiveness of the proposed method.

Assessment of biocompatibility and initial evaluation of genipin cross-linked elastin-like polypeptides in the treatment of an osteochondral knee defect in rabbits

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient β-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for β-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

Strokes are the fifth leading cause of death in the United States, and ~87% of strokes are ischemic, meaning that they are caused by occlusion of a cerebral artery or its branches. This occlusion results in a lack of oxygen reaching the tissue, increases in intracellular calcium and glutamate release, and the activation of inflammatory processes, which can cause additional tissue loss and worsen neurological condition. The nuclear factor kappa B (NF‐κB) cascade is an inflammatory pathway activated by many cytokines and inflammatory signals that are elevated following ischemic stroke, making it a central hub for post‐stroke inflammation. While there are current therapies to treat the occlusion causing an ischemic stroke, they do not treat the inflammation induced injury that occurs. We have designed a novel inhibitor of the NF‐κB cascade attached to the carrier protein elastin‐like polypeptide (ELP) as an adjuvant to current revascularization therapies. ELP is based on the sequence of human elastin, has a long plasma half‐life, is thermally responsive, and has a tunable size. We hypothesize that ELPs can be used to deliver our inhibitory peptide SynB1‐ELP‐p50i to the infarcted region after ischemic stroke, reduce inflammation, and spare brain tissue. To begin, BV2 microglia, C8‐D1A astrocytes, and SH‐SY5Y neurons were treated with rhodamine labeled SynB1‐ELP‐p50i for 24 hours and imaged with confocal microscopy to determine if the protein was able to enter the cells. SynB1‐ELP‐p50i entered the cells and was primarily localized to the cytoplasm. Next, using the middle cerebral artery occlusion (MCAO) model of ischemic stroke in spontaneously hypertensive rats (SHRs), biodistribution and pharmacokinetics were assessed following a bolus administration of 50 mg/kg rhodamine labeled SynB1‐ELP‐p50i, using two routes of administration (intravenous (IV, femoral vein) and intraarterial (IA, carotid catheter)). SynB1‐ELP‐p50i accumulated in the ischemic hemisphere at levels nearly 8 fold higher than in the contralateral hemisphere following MCAO (two‐way ANOVA F(1,12) = 63.08; Sidak’s p < 0.0001). There was no difference in intrabrain deposition between IV and IA routes. To assess the efficacy of SynB1‐ELP‐p50i (50 mg/kg, IV) to reduce infarct size and inflammation following MCAO, the brains were stained, and infarct size was measured using Image J. There was no effect of SynB1‐ELP‐p50i treatment on infarct size compared to saline controls, and the baseline mean arterial pressures were the same between treatment groups. qPCR analysis of downstream targets of the NF‐κB cascade are ongoing to determine if SynB1‐ELP‐p50i treatment affected the production of inflammatory cytokines following MCAO. Future studies will assess effects of SynB1‐ELP‐p50i treatment on behavior and neurological deficits following MCAO in SHRs.

Global meat consumption increased four-fold during the last fifty years, while population doubled1. Even if the increase in European meat consumption has slowed (currently 80 kg per capita, twice the world average), it is forecasted to increase by 10% more to 20302–4. The increase in meat eating is also nutritionally alarming as excessive consumption has been linked to health problems, such as coronary heart disease and certain cancers5. Fibrous, meat-like analogues are today commercially produced from soy, pea and wheat, utilizing an extruder to form a protein melt at high moisture content, high temperature and high pressure with subsequent active cooling on exit. A common denominator for the fibre formation in meat-analogues and plastics is that it is known how to produce the fibres but not exactly why they are formed. Consequently, it is still difficult to utilize the full potential of these techniques. The current hypothesis on the mechanisms responsible for the fibre formation contribute to understanding but are not sufficient to fully describe the formation and cannot be used to predict fibre formation ability of protein melts thus hampering the use of more sustainable protein sources. Overall, the hypotheses range from “physical”6–8, describing mechanisms in terms of fluid dynamics, heat transfer and phase separation, to “chemical” emphasizing the chemical interactions between protein chains or polymer crystallites. This contribution will focus on rheology of the protein melts, and especially on how to use state-of-the-art statistical analysis to determine the influence of temperature, protein and moisture content on rheological properties of the melts.

Tailoring ternary complexes of lactoferrin, EGCG, and α-lactalbumin by assembly sequence strategies: Structural characterization, assembly mechanism, and emulsification elucidation.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted inenrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

Elastin is the polymeric extracellular matrix protein responsible for the properties of extensibility and elastic recoil in tissues such as arterial blood vessels, lung parenchyma, and skin. Both tropoelastin (TE), the full-length monomeric form of elastin, and elastin-like polypeptides (ELPs), based on sequences and domain arrangements of TE, have the intrinsic ability to undergo organized self-assembly into network structures through a process of temperature-induced phase separation or coacervation. It has been suggested that this property plays a role in in vivo formation of the extracellular elastic matrix. In general, the temperature at which phase separation takes place has been taken as the measure of propensity for self-assembly. However, this phase separation is only the first step in a more complex, multistep process of network formation. We have previously shown that analysis of spectrophotometric data allows extraction of kinetic parameters describing both early (coacervation) and later (maturation) steps of the self-assembly process. Here, using a well-characterized ELP containing three hydrophobic domains flanking two cross-linking domains, we describe the effects of temperature, polypeptide concentration, and solution conditions on the kinetics of self-assembly, providing insights into possible mechanisms for the spontaneous organization of such ELPs into extended networks.

REV1, REV3, and REV7 are pivotal proteins in translesion DNA synthesis, which allows DNA synthesis even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases and function as inserter and extender polymerases in this process, respectively. REV7 interacts with both REV1 and REV3, acting as an adaptor that functionally links the two, although the structural basis of this collaboration remains unclear. Here, we show the crystal structure of the ternary complex, composed of the C-terminal domain of human REV1, REV7, and a REV3 fragment. The REV1 C-terminal domain adopts a four-helix bundle that interacts with REV7. A linker region between helices 2 and 3, which is conserved among mammals, interacts with the β-sheet of REV7. Remarkably, the REV7-binding interface is distinct from the binding site of DNA polymerase η or κ. Thus, the REV1 C-terminal domain might facilitate polymerase switching by providing a scaffold for both inserter and extender polymerases to bind. Our structure reveals the basis of DNA polymerase ζ (a complex of REV3 and REV7) recruitment to the stalled replication fork and provides insight into the mechanism of polymerase switching.

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.

Fiber Formation across the Bacterial Outer Membrane by the Chaperone/Usher Pathway