Peptide-Assembled Single-Chain Atomic Crystal Enhances Pluripotent Stem Cell Differentiation to Neurons.

Nanomaterials hybridized with biological components have widespread applications. among many candidates, peptides are attractive in that their peptide sequences can self-assemble with the surface of target materials with high specificity without perturbing the intrinsic properties of nanomaterials. Here, a 1D hybrid nanomaterial was developed through self-assembly of a designed peptide. A hexagonal coiled-coil motif geometrically matched to the diameter of the inorganic nanomaterial was fabricated, whose hydrophobic surface was wrapped along the axis of the hydrophobic core of the coiled coil. Our morphological and spectroscopic analyses revealed rod-shaped, homogeneous peptide-inorganic nanomaterial complexes. Culturing embryonic stem cells on surfaces coated with this peptide-assembled single-chain atomic crystal increased the growth and adhesion of the embryonic stem cells. The hybridized nanomaterial also served as an ECM for brain organoids, accelerating the maturation of neurons. New methods to fabricate hybrid materials through peptide assembly can be applied.

Sequence-dependent aggregation-prone conformations of islet amyloid polypeptide.

Amyloid proteins, which aggregate to form highly ordered structures, play a crucial role in various disease pathologies. Despite many previous studies on amyloid fibrils, which are an end product of protein aggregation, the structural characteristics of amyloid proteins in the early stage of aggregation and their related aggregation mechanism still remain elusive. The role of the amino acid sequence in the aggregation-prone structures of amyloid proteins at such a stage is not understood. Here, we have studied the sequence-dependent structural characteristics of islet amyloid polypeptide based on atomistic simulations and spectroscopic experiments. We show that the amino acid sequence determines non-bonded interactions that play a leading role in the formation of aggregation-prone conformations. Specifically, a single point mutation critically changes the population of aggregation-prone conformations, resulting in a change of the aggregation mechanism. Our simulation results were supported by experimental results suggesting that mutation affects the kinetics of aggregation and the structural characteristics of amyloid aggregates. Our study provides an insight into the role of sequence-dependent aggregation-prone conformations in the underlying mechanisms of amyloid aggregation.

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.

The Orientations of Large Aspect-Ratio Coiled-Coil Proteins Attached to Gold Nanostructures.

Methods for patterning biomolecules on a substrate at the single molecule level have been studied as a route to sensors with single-molecular sensitivity or as a way to probe biological phenomena at the single-molecule level. However, the arrangement and orientation of single biomolecules on substrates has been less investigated. Here, the arrangement and orientation of two rod-like coiled-coil proteins, cortexillin and tropomyosin, around patterned gold nanostructures is examined. The high aspect ratio of the coiled coils makes it possible to study their orientations and to pursue a strategy of protein orientation via two-point attachment. The proteins are anchored to the surfaces using thiol groups, and the number of cysteine residues in tropomyosin is varied to test how this variation affects the structure and arrangement of the surface-attached proteins. Molecular dynamics studies are used to interpret the observed positional distributions. Based on initial studies of protein attachment to gold post structures, two 31-nm-long tropomyosin molecules are aligned between the two sidewalls of a trench with a width of 68 nm. Because the approach presented in this study uses one of twenty natural amino acids, this method provides a convenient way to pattern biomolecules on substrates using standard chemistry.

Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.

Peptide-Drug Conjugate with Statistically Designed Transcellular Peptide for Psoriasis-Like Inflammation.

Peptide-drug conjugates (PDCs) are a promising class of drug delivery systems that utilize covalently conjugated carrier peptides with therapeutic agents. PDCs offer several advantages over traditional drug delivery systems including enhanced target engagement, improved bioavailability, and increased cell permeability. However, the development of efficient transcellular peptides capable of effectively transporting drugs across biological barriers remains an unmet need. In this study, physicochemical criteria based on cell-penetrating peptides are employed to design transcellular peptides derived from an antimicrobial peptides library. Among the statistically designed transcellular peptides (SDTs), SDT7 exhibits higher skin permeability, faster kinetics, and improved cell permeability in human keratinocyte cells compared to the control peptide. Subsequently, it is found that 6-Paradol (PAR) exhibits inhibitory activity against phosphodiesterase 4, which can be utilized for an anti-inflammatory PDC. The transcellular PDC (SDT7-conjugated with PAR, named TM5) is evaluated in mouse models of psoriasis, exhibiting superior therapeutic efficacy compared to PAR alone. These findings highlight the potential of transcellular PDCs (TDCs) as a promising approach for the treatment of inflammatory skin disorders.

Mechanistic insights into the anti-restenotic effects of HSP27 and HO1 modulated by reconstituted HDL on neointimal hyperplasia.

High-density lipoprotein (HDL) therapy has demonstrated beneficial effects in acute stroke and acute myocardial infarction models by reducing infarct size. In this study, we investigated the inhibitory effects of reconstituted HDL (rHDL) on neointimal hyperplasia and elucidated its underlying mechanism using a balloon injury rat model. Our finding revealed a significant 37% reduction in the intima to media ratio in the arteries treated with 80 mg/kg rHDL compared to those subjected to injury alone (p < 0.05), indicating a specific inhibition of neointimal hyperplasia. In vivo analysis further supported the positive effects of rHDL by demonstrating a reduction in smooth muscle cell (SMC) proliferation and an increase in endothelial cell (EC) proliferation. Additionally, rHDL treatment led to decreased infiltration of leukocytes and downregulated the expression of matrix metallopeptidase 9 (MMP9) in the neointimal area. Notably, rHDL administration resulted in decreased expression of VCAM1 and HIF1α, alongside increased expression of heme oxygenase 1 (HO1) and heat shock protein 27 (HSP27). Overexpression of HSP27 and HO1 effectively inhibited SMC proliferation. Moreover, rHDL-mediated suppression of injury-induced HIF1α coincided with upregulation of HSP27. Interestingly, HSP27 and HO1 had varying effects on the expression of chemokine receptors and rHDL did not exert significant effect on chemokine receptor expression in THP1 cells. These findings underscore the distinct roles of HSP27 and HO1 as potential regulatory factors in the progression of restenosis. Collectively, our study demonstrates that rHDL exerts a potent anti-neointimal hyperplasia effect by reducing leukocytes infiltration and SMC proliferation while promoting EC proliferation.

Supramolecular assembly of protein building blocks: from folding to function.

Several phenomena occurring throughout the life of living things start and end with proteins. Various proteins form one complex structure to control detailed reactions. In contrast, one protein forms various structures and implements other biological phenomena depending on the situation. The basic principle that forms these hierarchical structures is protein self-assembly. A single building block is sufficient to create homogeneous structures with complex shapes, such as rings, filaments, or containers. These assemblies are widely used in biology as they enable multivalent binding, ultra-sensitive regulation, and compartmentalization. Moreover, with advances in the computational design of protein folding and protein-protein interfaces, considerable progress has recently been made in the de novo design of protein assemblies. Our review presents a description of the components of supramolecular protein assembly and their application in understanding biological phenomena to therapeutics.

Effect of Secondary Structures on the Adsorption of Peptides onto Hydrophobic Solid Surfaces Revealed by SALDI-TOF and MD Simulations.

The adsorption of peptides and proteins on hydrophobic solid surfaces has received considerable research attention owing to their wide applications to biocompatible nanomaterials and nanodevices, such as biosensors and cell adhesion materials with reduced nanomaterial toxicity. However, fundamental understandings about physicochemical hydrophobic interactions between peptides and hydrophobic solid surfaces are still unknown. In this study, we investigate the effect of secondary structures on adsorption energies between peptides and hydrophobic solid surfaces via experimental and theoretical analyses using surface-assisted laser desorption/ionization-time-of-flight (SALDI-TOF) and molecular dynamics (MD) simulations. The hydrophobic interactions between peptides and hydrophobic solid surfaces measured via SALDI-TOF and MD simulations indicate that the hydrophobic interaction of peptides with random coil structures increased more than that of peptides with an α-helix structure when polar amino acids are replaced with hydrophobic amino acids. Additionally, our study sheds new light on the fundamental understanding of the hydrophobic interaction between hydrophobic solid surfaces and peptides that have diverse secondary structures.

Adherens junctions organize size-selective proteolytic hotspots critical for Notch signalling.

Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.