ABSTRACT
Assemblies of peptides and proteins through specific intermolecular interactions set the basis for macroscopic materials found in nature. Peptides provide easily tunable hydrogen-bonding interactions, which can lead to the formation of ordered structures such as highly stable ß-sheets that can form amyloid-like supramolecular peptide nanofibrils (PNFs). PNFs are of special interest, as they could be considered as mimics of various fibrillar structures found in nature. In their ability to serve as supramolecular scaffolds, they could mimic certain features of the extracellular matrix to provide stability, interact with pathogens such as virions, and transduce signals between the outside and inside of cells. Many PNFs have been reported that reveal rich bioactivities. PNFs supporting neuronal cell growth or lentiviral gene transduction have been studied systematically, and their material properties were correlated to bioactivities. However, the impact of the structure of PNFs, their dynamics, and stabilities on their unique functions is still elusive. Herein, we provide a microscopic view of the self-assembled PNFs to unravel how the amino acid sequence of self-assembling peptides affects their secondary structure and dynamic properties of the peptides within supramolecular fibrils. Based on sequence truncation, amino acid substitution, and sequence reordering, we demonstrate that peptide-peptide aggregation propensity is critical to form bioactive ß-sheet-rich structures. In contrast to previous studies, a very high peptide aggregation propensity reduces bioactivity due to intermolecular misalignment and instabilities that emerge when fibrils are in close proximity to other fibrils in solution. Our multiscale simulation approach correlates changes in biological activity back to single amino acid modifications. Understanding these relationships could lead to future material discoveries where the molecular sequence predictably determines the macroscopic properties and biological activity. In addition, our studies may provide new insights into naturally occurring amyloid fibrils in neurodegenerative diseases.
Subject(s)
Amyloid , Hydrophobic and Hydrophilic Interactions , Amyloid/chemistry , Peptides/chemistry , Protein Aggregates , Humans , Molecular Dynamics Simulation , Nanofibers/chemistry , Protein Structure, SecondaryABSTRACT
Retroviral gene delivery is the key technique for in vitro and ex vivo gene therapy. However, inefficient virion-cell attachment resulting in low gene transduction efficacy remains a major challenge in clinical applications. Adjuvants for ex vivo therapy settings need to increase transduction efficiency while being easily removed or degraded post-transduction to prevent the risk of venous embolism after infusing the transduced cells back to the bloodstream of patients, yet no such peptide system have been reported thus far. In this study, peptide amphiphiles (PAs) with a hydrophobic fatty acid and a hydrophilic peptide moiety that reveal enhanced viral transduction efficiency are introduced. The PAs form ß-sheet-rich fibrils that assemble into positively charged aggregates, promoting virus adhesion to the cell membrane. The block-type amphiphilic sequence arrangement in the PAs ensures efficient cell-virus interaction and biodegradability. Good biodegradability is observed for fibrils forming small aggregates and it is shown that via molecular dynamics simulations, the fibril-fibril interactions of PAs are governed by fibril surface hydrophobicity. These findings establish PAs as additives in retroviral gene transfer, rivalling commercially available transduction enhancers in efficiency and degradability with promising translational options in clinical gene therapy applications.
Subject(s)
Gene Transfer Techniques , Peptides , Humans , Peptides/chemistry , Genetic Therapy , Adjuvants, ImmunologicABSTRACT
Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.
Subject(s)
Killer Cells, Natural , T-Lymphocytes , Transduction, Genetic , Peptides , Immunotherapy, Adoptive/methodsABSTRACT
Gene therapy via retroviral vectors holds great promise for treating a variety of serious diseases. It requires the use of additives to boost infectivity. Amyloid-like peptide nanofibers (PNFs) were shown to efficiently enhance retroviral gene transfer. However, the underlying mode of action of these peptides remains largely unknown. Data-mining is an efficient method to systematically study structure-function relationship and unveil patterns in a database. This data-mining study elucidates the multi-scale structure-property-activity relationship of transduction enhancing peptides for retroviral gene transfer. In contrast to previous reports, we find that not the amyloid fibrils themselves, but rather µm-sized ß-sheet rich aggregates enhance infectivity. Specifically, microscopic aggregation of ß-sheet rich amyloid structures with a hydrophobic surface pattern and positive surface charge are identified as key material properties. We validate the reliability of the amphiphilic sequence pattern and the general applicability of the key properties by rationally creating new active sequences and identifying short amyloidal peptides from various pathogenic and functional origin. Data-mining-even for small datasets-enables the development of new efficient retroviral transduction enhancers and provides important insights into the diverse bioactivity of the functional material class of amyloids.
Subject(s)
Amyloidogenic Proteins , Data Mining , Reproducibility of Results , Databases, Factual , Peptides , RetroviridaeABSTRACT
Amyloid-like nanofibers from self-assembling peptides can promote viral gene transfer for therapeutic applications. Traditionally, new sequences are discovered either from screening large libraries or by creating derivatives of known active peptides. However, the discovery of de novo peptides, which are sequence-wise not related to any known active peptides, is limited by the difficulty to rationally predict structure-activity relationships because their activities typically have multi-scale and multi-parameter dependencies. Here, we used a small library of 163 peptides as a training set to predict de novo sequences for viral infectivity enhancement using a machine learning (ML) approach based on natural language processing. Specifically, we trained an ML model using continuous vector representations of the peptides, which were previously shown to retain relevant information embedded in the sequences. We used the trained ML model to sample the sequence space of peptides with 6 amino acids to identify promising candidates. These 6-mers were then further screened for charge and aggregation propensity. The resulting 16 new 6-mers were tested and found to be active with a 25% hit rate. Strikingly, these de novo sequences are the shortest active peptides for infectivity enhancement reported so far and show no sequence relation to the training set. Moreover, by screening the sequence space, we discovered the first hydrophobic peptide fibrils with a moderately negative surface charge that can enhance infectivity. Hence, this ML strategy is a time- and cost-efficient way for expanding the sequence space of short functional self-assembling peptides exemplified for therapeutic viral gene delivery.
Subject(s)
Nanofibers , Peptides , Amino Acid Sequence , Peptides/chemistry , AmyloidABSTRACT
Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.
Subject(s)
COVID-19 , Zika Virus Infection , Zika Virus , Humans , Peptides , Amyloid/chemistry , Anti-Bacterial Agents/pharmacology , HemoglobinsABSTRACT
Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.
Subject(s)
Amyloid , Peptides , Cricetinae , Animals , Amyloid/chemistry , Amyloid/metabolism , CHO Cells , Cricetulus , Protein Structure, SecondaryABSTRACT
Gradients of bioactive molecules play a crucial role in various biological processes like vascularization, tissue regeneration, or cell migration. To study these complex biological systems, it is necessary to control the concentration of bioactive molecules on their substrates. Here, we created a photochemical strategy to generate gradients using amyloid-like fibrils as scaffolds functionalized with a model epitope, that is, the integrin-binding peptide RGD, to modulate cell adhesion. The self-assembling ß-sheet forming peptide (CKFKFQF) was connected to the RGD epitope via a photosensitive nitrobenzyl linker and assembled into photoresponsive nanofibrils. The fibrils were spray-coated on glass substrates and macroscopic gradients were generated by UV-light over a centimeter-scale. We confirmed the gradient formation using matrix-assisted laser desorption ionization mass spectroscopy imaging (MALDI-MSI), which directly visualizes the molecular species on the surface. The RGD gradient was used to instruct cells. In consequence, A549 adapted their adhesion properties in dependence of the RGD-epitope density.
Subject(s)
Amyloid , Oligopeptides , A549 Cells , Cell Adhesion , Cell Movement , Humans , PeptidesABSTRACT
Therapeutic viral gene delivery is an emerging technology which aims to correct genetic mutations by introducing new genetic information to cells either to correct a faulty gene or to initiate cell death in oncolytic treatments. In recent years, significant scientific progress has led to several clinical trials resulting in the approval of gene therapies for human treatment. However, successful therapies remain limited due to a number of challenges such as inefficient cell uptake, low transduction efficiency (TE), limited tropism, liver toxicity and immune response. To adress these issues and increase the number of available therapies, additives from a broad range of materials like polymers, peptides, lipids, nanoparticles, and small molecules have been applied so far. The scope of this review is to highlight these selected delivery systems from a materials perspective.