Resilin-mimetics as a smart biomaterial platform for biomedical applications

Abstract

Intrinsically disordered proteins have dramatically changed the structure-function paradigm of proteins in the 21^st century. Resilin is a native elastic insect protein, which features intrinsically disordered structure, unusual multi-stimuli responsiveness and outstanding resilience. Advances in computational techniques, polypeptide synthesis methods and modular protein engineering routines have led to the development of novel resilin-like polypeptides (RLPs) including modular RLPs, expanding their applications in tissue engineering, drug delivery, bioimaging, biosensors, catalysis and bioelectronics. However, how the responsive behaviour of RLPs is encoded in the amino acid sequence level remains elusive. This review summarises the milestones of RLPs, and discusses the development of modular RLP-based biomaterials, their current applications, challenges and future perspectives. A perspective of future research is that sequence and responsiveness profiling of RLPs can provide a new platform for the design and development of new modular RLP-based biomaterials with programmable structure, properties and functions.

Fig. 1. Timeline of key discoveries and milestones of resilin and RLP-based biomaterials.

Description of illustrations (in the order of year): 1960—locust-wing hinge showing resilin, 1964—covalent dityrosine crosslinks in native resilin, 1966—native uncrosslinked pro-resilin synthesized by epidermal cells into the subcuticular space, 2001—full-length resilin (fruit fly) showing the three exon regions with respective sequence length, 2005—RLPs synthesis by recombinant technology, 2009—domain selection and design of modular RLP, 2011 (top)—RLP (antigen) and RLP-antibody interaction for bioimaging, 2011 (bottom)—plot (temperature versus hydrodynamic diameter) showing both upper critical solution temperature (UCST) and lower critical solution temperature (LCST), dual-phase behaviour of RLPs, 2012—structural conformation of full-length resilin to stress, 2013—crosslinked network between RLPs (blue) and four-arm polyethylene glycol (PEG; green) hybrid hydrogel, 2015—plot (temperature versus ultra-violet absorbance) showing soluble to insoluble transition of RLPs in aqueous solution and 2018—resilin gene expression in plants.

Fig. 2. Evaluation of functional intrinsic disorder propensity and interactivity of D. melanogaster pro-resilin: full-length resilin (UniProt ID: Q9V7U0).

a Functional disorder profile generated by D²P² platform (http://d2p2.pro/). The nine different coloured bars represent location of disordered regions found by different disorder predictors (Espritz-D, Espritz-X, etc.). The green bar corresponds to disordered regions by consensus, and shows the predicted disorder agreement between these nine predictors. The yellow bar corresponds to disorder-based binding sites known as molecular recognition features (MoRFs). b Interactability of resilin analysed (using the medium confidence level of 0.4) by STRING platform (http://string-db.org/cgi/). The coloured nodes represent query protein (red) and first shell of protein interactors (with gene name or ID), where empty and filled nodes represent proteins of unknown 3D structure and some known or predicted 3D structure, respectively. Protein–protein interaction is represented by differently coloured lines, where green line represents neighbourhood evidence, black line represents co-expression evidence, purple line represents experimental evidence and light blue line represent database evidence.

Fig. 3. Schematic of key steps involved in modular RLPs synthesis.

The domain selection step involves the selection of peptide domains of desired functions, the design step involves the arrangement of the intended amino acid sequence and encoding genetic sequence, the incorporation step involves the construction of a genetic vector and their transfection into a host organism, the expression involves induced expression of the intended modular RLPs in host organism, and purification step involves extraction of pure polypeptides by chromatographic and/or non-chromatographic (cold-coacervation) methods.

Fig. 4. Hallmarks of RLPs, development of modular RLP-based materials and their structure–property relationship.

a Summary of RLPs, secondary domains and other materials applied for development of modular RLP-based nanostructures and hybrids. The amino acid sequences of RLP repeats and secondary domains are presented as a single letter code. UCST upper critical solution temperature, LCST lower critical solution temperature, ELP elastin-like polypeptide, SLP silk-like polypeptide, CLP collagen-like polypeptide, MMP matrix metalloproteinase cleavable peptide, HBD heparin-binding domain, BMP bone morphogenetic peptide, QK vascular endothelial growth factor-mimicking domain, CBM cellulose-binding module, HFBI hydrophobin protein domain and LCD lysine crosslinking domain. b Schematics of dual-phase transition behaviour and some of the structure–property relationship of RLP- and modular RLP-based systems^,.

Fig. 5. Schematic of design consideration for RLP-based smart nanoparticle and hydrogel systems to enable immunomodulation.

Design elements, including size, shape, molecular recognition, hydrophobicity, surface charge and stiffness of RLP-based biomaterials can modulate their immune cell (dendritic cells, macrophages, T cells, B cells, etc.) interaction and molecular response.