Cation Triggered Self-Assembly of α-Lactalbumin Nanotubes.

Metal ions play a dual role in biological systems. Although they actively participate in vital life processes, they may contribute to protein aggregation and misfolding and thus contribute to development of diseases and other pathologies. In nanofabrication, metal ions mediate the formation of nanostructures with diverse properties. Here, we investigated the self-assembly of α-lactalbumin into nanotubes induced by coordination with metal ions, screened among the series Mn2+, Co2+, Ni2+, Zn2+, Cd2+, and Au3+. Our results revealed that the affinity of metal ions toward hydrolyzed α-lactalbumin peptides not only impacts the kinetics of nanotube formation but also influences their length and rigidity. These findings expand our understanding of supramolecular assembly processes in protein-based materials and pave the way for designing novel materials such as metallogels in biochip and biosensor applications.

Bioengineered "Molecular Glue"-Mediated Tumor-Specific Cascade Nanoreactors with Self-Destruction Ability for Enhanced Precise Starvation/Chemosynergistic Tumor Therapy.

The ordered and directed functionalization of targeting elements on the surface of nanomaterials for precise tumor therapy remains a challenge. To address the above problem, herein, we adopted a materials-based synthetic biotechnology strategy to fabricate a bioengineered fusion protein of materials-binding peptides and targeting elements, which can serve as a "molecular glue" to achieve a directional and organized assembly of targeting biological macromolecules on the surface of nanocarriers. The hypoxia microenvironment of solid tumors inspired the rapid development of starvation/chemosynergistic therapy; however, the unsatisfied spatiotemporal specific performance hindered its further development in precise tumor therapy. As a proof of concept, a bioengineered fusion protein containing a dendritic mesoporous silicon (DMSN)-binding peptide, and a tumor-targeted and acidity-decomposable ferritin heavy chain 1 (FTH1), was constructed by fusion expression and further assembled on the surface of DMSN companying with the insertion of hypoxia-activated prodrug tirapazamine (TPZ) and glucose oxidase (GOX) to establish a nanoreactor for precise starvation/chemosynergistic tumor therapy. In this context, the as-prepared therapeutic nanoreactors revealed obvious tumor-specific accumulation and an endocytosis effect. Next, the acidic tumor microenvironment triggered the structural collapse of FTH1 and the subsequent release of GOX and TPZ, in which GOX-mediated catalysis cut off the nutrition supply to realize starvation therapy based on the consumption of endogenous glucose and further provided an exacerbated hypoxia environment for TPZ in situ activation to initiate tumor chemotherapy. More significantly, the presence of "molecular glue" elevated the tumor-targeting capacity of nanoreactors and further enhanced the starvation/chemosynergistic therapeutic effect remarkably, suggesting that such a strategy provided a solution for the functionality of nanomaterials and facilitated the design of novel targeting nanomedicines. Overall, this study highlights materials-binding peptides as a new type of "molecular glue" and opens new avenues for designing and exploring active biological materials for biological functions and applications.

Salt-Inducing Assembly Polymorphism Strategy for Cytotoxicity Differentiation of Phenol-Soluble Modulin α3 Assemblies.

Phenol-soluble modulin α3 (PSMα3) can self-assemble into fibrous assemblies with a unique "cross-α" sheet structure, which serves as a key virulence factor in the infection of Staphylococcus aureus. However, the structure-cytotoxicity relationships of PSMα3 still remain elusive. Herein, we utilized the strategy of salt-inducing assembly polymorphism to controllably prepare three PSMα3 assemblies with morphological and structural distinctions, including amorphous aggregates (AAs), rigid fibrils (RFs), and oligomers/curvilinear fibrils (OCFs), which provided a convincing method to facilitate the structure-cytotoxicity investigation of PSMα3 assemblies. Our results affirmed that amyloid fibrillation was essential for the enhancement of PSMα3 cytotoxicity, which was proved based on the evidence that RFs and OCFs both triggered more obvious cytotoxicity than AAs. Furthermore, our study also demonstrated that the cytotoxicity was severely dependent on the size and structure of PSMα3 fibrils. In detail, smaller OCFs rich in α-helices exhibited stronger virulence than RFs with larger sizes and low α-helical contents. The cytotoxicity caused by such fibrils was achieved via a membrane-disrupting mechanism, in which RFs and OCFs might be prone to membrane thinning and perforation, respectively. This strategy of salt-inducing PSMα3 assembly polymorphism facilitated the comprehension of the relationship between the characteristics of PSMα3 assemblies and their cytotoxicity and was also helpful to understanding the intrinsic assembly mechanism of the PSMα3.

Sappanwood-derived polyphenolic antidote of amyloidal toxins achieved detoxification via inhibition/reversion of amyloidal fibrillation.

The formidable virulence of methicillin-resistant staphylococcus aureus (MRSA) have thrown great challenges to biomedicine, which mainly derives from their autocrine phenol-soluble modulins (PSMs) toxins, especially the most toxic member termed phenol-soluble modulins α3 (PSMα3). PSMα3 cytotoxicity is attributed to its amyloidal fibrillation and subsequent formation of cross-α sheet fibrils. Inspired by the multiple biological activity of Sappanwood, herein, we adopted brazilin, a natural polyphenolic compound originated from Caesalpinia sappan, as a potential antidote of PSMα3 toxins, and attempted to prove that the regulation of PSMα3 fibrillation was an effective alexipharmic way for MRSA infections. In vitro results revealed that brazilin suppressed PSMα3 fibrillation and disassembled preformed amyloidal fibrils in a dose-dependent manner, in which molar ratio (brazilin: PSMα3) of efficient inhibition and disassembly were both 1:1. These desired regulations dominated by brazilin benefited from its bonding to core fibrils-forming residues of PSMα3 monomers urged by hydrogen bonding and pi-pi stacking, and such binding modes facilitated brazilin-mediated inhibition or disruption of interactions between neighboring PSMα3 monomers. In this context, these inhibited and disassembled PSMα3 assemblies could not easily insert into cell membrane and subsequent penetration, and thus alleviating the membrane disruption, cytoplasmic leakage, and reactive oxygen species (ROS) generation in normal cells. As such, brazilin dramatically decreased the cytotoxicity borne by toxic PSMα3 fibrils. In addition, in vivo experiments affirmed that brazilin relieved the toxicity of PSMα3 toxins and thus promoted the skin wound healing of mice. This study provides a new antidote of PSMα3 toxins, and also confirms the feasibility of the assembly-regulation strategy in development of antidotes against supramolecular fibrillation-dependent toxins.

Rational Biological Interface Engineering: Amyloidal Supramolecular Microstructure-Inspired Hydrogel.

Amyloidal proteins, which are prone to form fibrillar and ordered aggregates in vivo and in vitro, underlie the mechanism for neurodegenerative disorders and also play essential functions in the process of life. Amyloid fibrils typically adopt a distinctive ß-sheet structure, which renders them with inherent extracellular matrix (ECM)-mimicking properties, such as powerful mechanical strength, promising adhesion, and antibacterial activity. Additionally, amyloidal proteins are a category of programmable self-assembled macromolecules, and their assembly and consequent nanostructure can be manipulated rationally. The above advantages motivate researchers to investigate the potential of amyloidal proteins as a novel type of hydrogel material. Currently, the amyloid-inspired hydrogel has become an emerging area and has been widely applied in a variety of biomedical fields, such as tissue repair, cell scaffolds, and drug delivery. In this review, we focus on the discussion of molecular mechanisms underlying the hydrogenation of amyloidal proteins, and introduce the advances achieved in biomedical applications of amyloid-inspired hydrogels.

Binding Peptide-Guided Immobilization of Lipases with Significantly Improved Catalytic Performance Using Escherichia coli BL21(DE3) Biofilms as a Platform.

Developing novel immobilization methods to maximize the catalytic performance of enzymes has been a permanent pursuit of scientific researchers. Engineered Escherichia coli biofilms have attracted great concern as surface display platforms for enzyme immobilization. However, current biological conjugation methods, such as the SpyTag/SpyCatcher tagging pair, that immobilize enzymes onto E. coli biofilms seriously hamper enzymatic performance. Through phage display screening of lipase-binding peptides (LBPs) and co-expression of CsgB (nucleation protein of curli nanofibers) and LBP2-modified CsgA (CsgALBP2, major structural subunit of curli nanofibers) proteins, we developed E. coli BL21::ΔCsgA-CsgB-CsgALBP2 (LBP2-functionalized) biofilms as surface display platforms to maximize the catalytic performance of lipase (Lip181). After immobilization onto LBP2-functionalized biofilm materials, Lip181 showed increased thermostability, pH, and storage stability. Surprisingly, the relative activity of immobilized Lip181 increased from 8.43 to 11.33 U/mg through this immobilization strategy. Furthermore, the highest loading of lipase on LBP2-functionalized biofilm materials reached up to 27.90 mg/g of wet biofilm materials, equivalent to 210.49 mg/g of dry biofilm materials, revealing their potential as a surface with high enzyme loading capacity. Additionally, immobilized Lip181 was used to hydrolyze phthalic acid esters, and the hydrolysis rate against dibutyl phthalate was up to 100%. Thus, LBP2-mediated immobilization of lipases was demonstrated to be far more advantageous than the traditional SpyTag/SpyCatcher strategy in maximizing enzymatic performance, thereby providing a better alternative for enzyme immobilization onto E. coli biofilms.

Monomer-targeting affinity peptide inhibitors of amyloid with no self-fibrillation and low cytotoxicity.

We utilized solution-phase biopanning to obtain a de novo peptide (LA12) that specifically bound to the core region of the human amylin monomer. LA12 stabilized the random coil conformation of amylin to suppress aggregation in a dose-dependent manner with the highest suppression effect of 78% and reduced the cytotoxicity of amylin.