Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa)n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502GFPGER507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI.

Mapping the Effect of Gly Mutations in Collagen on α2ß1 Integrin Binding.

The replacement of one Gly in the essential repeating tripeptide sequence of the type I collagen triple helix results in the dominant hereditary bone disorder osteogenesis imperfecta. The mechanism leading to pathology likely involves misfolding and autophagy, although it has been hypothesized that some mutations interfere with known collagen interactions. Here, the effect of Gly replacements within and nearby the integrin binding GFPGER sequence was investigated using a recombinant bacterial collagen system. When a six-triplet human type I collagen sequence containing GFPGER was introduced into a bacterial collagen-like protein, this chimeric protein bound to integrin. Constructs with Gly to Ser substitutions within and nearby the inserted human sequence still formed a trypsin-resistant triple helix, suggesting a small local conformational perturbation. Gly to Ser mutations within the two Gly residues in the essential GFPGER sequence prevented integrin binding and cell attachment as predicted from molecular dynamics studies of the complex. Replacement of Gly residues C-terminal to GFPGER did not affect integrin binding. In contrast, Gly replacements N-terminal to the GFPGER sequence, up to four triplets away, decreased integrin binding and cell adhesion. This pattern suggests either an involvement of the triplets N-terminal to GFPGER in initial binding or a propagation of the perturbation of the triple helix C-terminal to a mutation site. The asymmetry in biological consequences relative to the mutation site may relate to the observed pattern of osteogenesis imperfecta mutations near the integrin binding site.

Fibrous proteins: At the crossroads of genetic engineering and biotechnological applications.

Fibrous proteins, such as silk, elastin and collagen are finding broad impact in biomaterial systems for a range of biomedical and industrial applications. Some of the key advantages of biosynthetic fibrous proteins compared to synthetic polymers include the tailorability of sequence, protein size, degradation pattern, and mechanical properties. Recombinant DNA production and precise control over genetic sequence of these proteins allows expansion and fine tuning of material properties to meet the needs for specific applications. We review current approaches in the design, cloning, and expression of fibrous proteins, with a focus on strategies utilized to meet the challenges of repetitive fibrous protein production. We discuss recent advances in understanding the fundamental basis of structure-function relationships and the designs that foster fibrous protein self-assembly towards predictable architectures and properties for a range of applications. We highlight the potential of functionalization through genetic engineering to design fibrous protein systems for biotechnological and biomedical applications.

Definition of the native and denatured type II collagen binding site for fibronectin using a recombinant collagen system.

Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.

Molecular basis for integrin adhesion receptor binding to p21-activated kinase 4 (PAK4).

Integrin adhesion receptors provide links between extracellular ligands and cytoplasmic signaling. Multiple kinases have been found to directly engage with integrin ß tails, but the molecular basis for these interactions remain unknown. Here, we assess the interaction between the kinase domain of p21-activated kinase 4 (PAK4) and the cytoplasmic tail of integrin ß5. We determine three crystal structures of PAK4-ß5 integrin complexes and identify the PAK-binding site. This is a region in the membrane-proximal half of the ß5 tail and confirmed by site-directed mutagenesis. The ß5 tail engages the kinase substrate-binding groove and positions the non-phosphorylatable integrin residue Glu767 at the phosphoacceptor site. Consistent with this, integrin ß5 is poorly phosphorylated by PAK4, and in keeping with its ability to occlude the substrate-binding site, weakly inhibits kinase activity. These findings demonstrate the molecular basis for ß5 integrin-PAK4 interactions but suggest modifications in understanding the potential cellular role of this interaction.

Toxicological assessment and food allergy of silk fibroin derived from Bombyx mori cocoons.

Recent studies have demonstrated silk fibroin protein's (SF) ability to extend the shelf life of foods by mitigating the hallmarks of spoilage, namely oxidation and dehydration. Due to the potential for this protein to become more widespread, its safety was evaluated comprehensively. First, a bacterial reverse mutation test (Ames test) was conducted in five bacterial strains. Second, an in vivo erythrocyte test was conducted with Sprague Dawley rats at doses up to 1,000mg/kg-bw/day. Third, a range-finder study was conducted with Sprague Dawley rats at the highest consumption amount given solubility and oral gavage volume constrains (500mg/kg-bw/day). Fourth, a 28-day sub-chronic study in Sprague Dawley rats was conducted with the high dose set at 500mg/kg-bw/day, as limited by solubility of the protein in a single-gavage per-day study. Fifth, an in vitro pepsin digestion assay was performed to assess the potential for protein allergenicity. Sixth, allergenic potential was further assessed using liquid chromatography-mass spectroscopy for detection of allergenic insect proteins. Seventh, the SF protein sequences were subjected to bioinformatic analyses. Together, these studies raise no mutagenic, genotoxic, toxicological, or allergenic concerns with the oral consumption of silk fibroin.

Synergistic effect of exogeneous and endogeneous electrostimulation on osteogenic differentiation of human mesenchymal stem cells seeded on silk scaffolds.

Bioelectrical regulation of bone fracture healing is important for many cellular events such as proliferation, migration, and differentiation. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk scaffolds in response to different modes of electrostimulation (e.g., exogeneous and/or endogeneous). Endogeneous electrophysiology was altered through the use of monensin (10 nM) and glibenclamide (10 µM), along with external electrostimulation (60 kHz; 100-500 mV). Monensin enhanced the expression of early osteogenic markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX-2). When exogeneous electrostimulation was combined with glibenclamide, more mature osteogenic marker upregulation based on bone sialoprotein expression (BSP) and mineralization was found. These results suggest the potential to exploit both exogeneous and endogeneous biophysical control of cell functions towards tissue-specific goals.

Formation of multimers of bacterial collagens through introduction of specific sites for oxidative crosslinking.

A range of non-animal collagens has been described, derived from bacterial species, which form stable triple-helical structures without the need for secondary modification to include hydroxyproline in the sequence. The non-animal collagens studied to date are typically smaller than animal interstitial collagens, around one quarter the length and do not pack into large fibrillar aggregates like those that are formed by the major animal interstitial collagens. A consequence of this for biomedical products is that fabricated items, such as collagen sponges, are not as mechanically and dimensionally stable as those of animal collagens. In the present study, we examined the production of larger, polymeric forms of non-animal collagens through introduction of tyrosine and cysteine residues that can form selective crosslinks through oxidation. These modifications allow the formation of larger aggregates of the non-animal collagens. When Tyr residues were incorporated, gels were obtained. And with Cys soluble aggregates were formed. These materials can be formed into sponges that are more stable than those formed without these modifications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2369-2376, 2016.

Bioengineered silk gene delivery system for nuclear targeting.

Gene delivery research has gained momentum with the use of lipophilic vectors that mimic viral systems to increase transfection efficiency. Maintaining cell viability with these systems remains a major challenge. Therefore, biocompatible biopolymers that are designed by combining non-immunological viral mimicking components with suitable carrier are explored to address these limitations. In the present study, dragline silk recombinant proteins are modified with DNA condensing units and the proton sponge endosomal escape pathway is utilized for enhanced delivery. Transfection efficiency in a COS-7 cell line is enhanced compared to lipofectamine and polyethyleneimine (PEI), as is cell viability.

Fabrication and functionalization of hydrogels through "click" chemistry.

Hydrogels are crosslinked polymeric materials that play a vital role in many biomedical areas such as drug delivery, sensor technology, and tissue engineering. Increasing demand of these materials for such advanced applications has necessitated the development of hydrogels with complex chemical compositions such as incorporating small molecules and biomolecules that provide the functional attributes. This Focus Review highlights the tremendous impact of click chemistry on the design, synthesis, and functionalization of hydrogels. The high efficiency and fidelity of the click reactions have enabled rapid and modular synthesis of hydrogels with near-ideal network structures. Efficient incorporation of biomolecular building blocks, such as peptide sequences either during or after the fabrication of hydrogels, have been achieved through various click reactions. Utilization of these efficient reactions has led to the fabrication of many stimuli-responsive or 'smart' hydrogels in recent years.