Fusion Peptide Research Articles

Plant-Produced Viral Nanoparticles as a Functionalized Catalytic Support for Metabolic Engineering.

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed.

A comprehensive investigation of the medicinal efficacy of antimicrobial fusion peptides expressed in probiotic bacteria for the treatment of pan drug-resistant (PDR) infections.

The present work aimed to examine the intracellular antibacterial efficacy of Recombinant Lactobacillus acidophilus/antimicrobial peptides (AMPs) Melittin and Alyteserin-1a, specifically targeting Gram-negative bacteria. The first assessment was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Recombinant L. acidophilus/AMPs versus Gram-negative and Gram-positive bacteria. In addition, the researchers examined the in vitro viability and safety of AMPs generated by L. acidophilus. The experiments included exposing the AMPs to elevated temperatures, proteases, cationic salts at physiological levels, and specific pH settings. The safety aspect was evaluated using hemolytic analysis utilizing sheep erythrocytes; cytotoxicity assays employing cell lines, and experiments on beneficial gut lactobacilli. An experiment was done using a time-kill method to assess the intracellular antibacterial efficacy of Recombinant L. acidophilus/AMPs compared to pathogenic varieties in HEp-2 cells. Previous investigations have shown that the MBC levels of recombinant L. acidophilus/AMPs were consistently two to four times higher than the equivalent MIC values when evaluated versus Gram-negative bacteria. Furthermore, the stability of the Recombinant L. acidophilus/AMPs showed variability when exposed to elevated temperatures (70 and 90℃), treated with protease enzymes (proteinase K, lysozyme), exposed to higher concentrations of physiological salts (150mM NaCl and 2mM MgCl2), and varying pH levels (ranging from 4.0 to 9.0). The recombinant L. acidophilus/AMPs are non-hemolytic towards sheep erythrocytes, exhibit little cytotoxicity in RAW 264.7 and HEp-2 cells, and are considered safe when compared to beneficial gut lactobacilli. The research examined the intracellular bacteriostatic effects of recombinant L. acidophilus/AMPs on Gram-negative bacteria inside HEp-2 cells. Nevertheless, no notable bactericidal impact was seen on Gram-positive bacteria (P > 0.05). The research shows that recombinant L. acidophilus/AMPs, namely (L. acidophilus/melittin/Alyteserin-1a) as the focus of the investigation, effectively eliminate Gram-negative bacteria. Therefore, more investigation is necessary to elaborate on these discoveries.

Degron-Controlled Protein Degradation in Escherichia coli: New Approaches and Parameters.

Protein degron tags have proven to be uniquely useful for the characterization of gene function. Degrons can mediate quick depletion, usually within minutes, of a protein of interest, allowing researchers to characterize cellular responses to the loss of function. To develop a general-purpose degron tool in Escherichia coli, we sought to build upon a previously characterized system of SspB-dependent inducible protein degradation. For this, we created a family of expression vectors containing a destabilized allele of SspB, capable of a rapid and nearly perfect "off-to-on" induction response. Using this system, we demonstrated excellent control over several DNA metabolism enzymes. However, other substrates did not respond to degron tagging in such an ideal manner, indicating the apparent limitations of SspB-dependent systems. Several degron-tagged proteins were degraded too slowly to be completely depleted during active growth, whereas others appeared to be completely refractory to degron-promoted degradation. Thus, only a minority of our, admittedly biased, selection of degron substrates proved to be amenable to efficient SspB-catalyzed degradation. We also uncovered an apparent stalling and/or disengagement of ClpXP from a degron-tagged allele of beta-galactosidase (beta-gal). While a degron-containing fusion peptide attached to the carboxy-terminus of beta-gal was degraded quantitatively, no reductions in beta-gal activity or concentration were detected, demonstrating an apparently novel mechanism of protease resistance. We conclude that substrate-dependent effects of the SspB system present a continued challenge to the widespread adoption of this degron system. For substrates that prove to be degradable, we provide a series of titratable SspB-expression vehicles.

Reversal of the fusion catalytic role of SARS-CoV-2 fusion peptide by restricting the N terminus: Implications to tackle viral infection

Reversal of the fusion catalytic role of SARS-CoV-2 fusion peptide by restricting the N terminus: Implications to tackle viral infection

Lactoferricin B Combined with Antibiotics Exhibits Leukemic Selectivity and Antimicrobial Activity.

The fusion of penetrating peptides (PPs), e.g., cell penetration peptides (CPPs) or antimicrobial peptides (AMPs), together with antimicrobial agents is an expanding research field. Specific AMPs, such as lactoferricin B (LfcinB), have demonstrated strong antibacterial, antifungal, and antiparasitic activity, as well as valuable anticancer activity, proving beneficial in the development of anticancer conjugates. The resulting conjugates offer potential dual functionality, acting as both an anticancer and an antimicrobial agent. This is especially necessary in cancer treatment, where microbial infections pose a critical risk. Leukemic cells frequently exhibit altered outer lipid membranes compared to healthy cells, making them more sensitive to compounds that interfere with their membrane. In this study, we revisited and reanalyzed our earlier research on LfcinB and its conjugates. Furthermore, we carried out new experiments with a specific focus on cell proliferation, changes in membrane asymmetric phosphatidylserine location, intracellular reactive oxygen species (ROS) generation, mitochondrial functions, and in vitro bacterial topoisomerase inhibition.

The fusion peptide of the influenza virus spike mediates aggregation, poration, and membrane fusion in membranes with different compositions

The fusion peptide of the influenza virus spike mediates aggregation, poration, and membrane fusion in membranes with different compositions

Insertion and anchoring of HIV-1 fusion peptide into complex membrane mimicking the human T cell

Insertion and anchoring of HIV-1 fusion peptide into complex membrane mimicking the human T cell

Fusion peptide dynamics during receptor-induced conformational changes in the HIV-1 envelope

Fusion peptide dynamics during receptor-induced conformational changes in the HIV-1 envelope

Membrane composition affects binding and insertion of the influenza virus fusion peptide

Membrane composition affects binding and insertion of the influenza virus fusion peptide

Cleavage-Activation of Respiratory Viruses - Half a Century of History from Sendai Virus to SARS-CoV-2.

Many viruses require the cleavage-activation of membrane fusion proteins by host proteases in the course of infection. This knowledge is based on historical studies of Sendai virus in the 1970s. From the 1970s to the 1990s, avian influenza virus and Newcastle disease virus were studied, showing a clear link between virulence and the cleavage-activation of viral membrane fusion proteins (hemagglutinin and fusion proteins) by host proteases. In these viruses, cleavage of viral membrane fusion proteins by furin is the basis for their high virulence. Subsequently, from the 2000s to the 2010s, the importance of TMPRSS2 in activating the membrane fusion proteins of various respiratory viruses, including seasonal influenza viruses, was demonstrated. In late 2019, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) emerged and caused a pandemic. The virus continues to mutate, producing variants that have caused global pandemics. The spike protein of SARS-CoV-2 is characterized by two cleavage sites, each of which is cleaved by furin and TMPRSS2 to achieve membrane fusion. SARS-CoV-2 variants exhibit altered sensitivity to these proteases. Thus, studying the cleavage-activation of membrane fusion proteins by host proteases is critical for understanding the ongoing pandemic and developing countermeasures against it.

Recombinant Antibody Fragments for Immunotherapy of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. Multiple genetic and environmental factors leading to progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SN) and consequent depletion of dopamine were described. Current clinical approaches, such as dopamine replacement or deep brain stimulation using surgically implanted probes, provide symptomatic relief but cannot modify disease progression. Therefore, disease-modifying therapeutic tools are urgently needed. Immunotherapy approaches, including passive transfer of protective antibodies and their fragments, have shown therapeutic efficacy in several animal models of neurodegenerative diseases, including PD. Recombinant antibody fragments are promising alternatives to conventional full-length antibodies. Modern computational approaches and molecular biology tools, directed evolution methodology, and the design of tissue-penetrating fusion peptides allowed for the development of recombinant antibody fragments with superior specificity and affinity, reduced immunogenicity, the capacity to target hidden epitopes and cross the blood-brain barrier (BBB), higher solubility and stability, the ability to refold after heat denaturation, and inexpensive large-scale production. In addition, antibody fragments do not induce microglia Fcγ receptor (FcγR)-mediated proinflammatory response and tissue damage in the central nervous system (CNS), because they lack the Fc portion of the immunoglobulin molecule. In the present review, we summarized data on recombinant antibody fragments evaluated as immunotherapeutics in preclinical models of PD and discussed their potential for developing therapeutic and preventive protocols for patients with PD.

Combined biolistic and cell penetrating peptide delivery for the development of scalable intradermal DNA vaccines

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET‑gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery.

A Mechanistic Understanding of the Modes of Ca2+ Ion Binding to the SARS-CoV-1 Fusion Peptide and Their Role in the Dynamics of Host Membrane Penetration.

The SARS-CoV-1 spike glycoprotein contains a fusion peptide (FP) segment that mediates the fusion of the viral and host cell membranes. Calcium ions are thought to position the FP optimally for membrane insertion by interacting with negatively charged residues in this segment (E801, D802, D812, E821, D825, and D830); however, which residues bind to calcium and in what combinations supportive of membrane insertion are unknown. Using biological assays and molecular dynamics studies, we have determined the functional configurations of FP-Ca2+ binding that likely promote membrane insertion. We first individually mutated the negatively charged residues in the SARS CoV-1 FP to assay their roles in cell entry and syncytia formation, finding that charge loss in the D802A or D830A mutants greatly reduced syncytia formation and pseudoparticle transduction of VeroE6 cells. Interestingly, one mutation (D812A) led to a modest increase in cell transduction, further indicating that FP function likely depends on calcium binding at specific residues and in specific combinations. To interpret these results mechanistically and identify specific modes of FP-Ca2+ binding that modulate membrane insertion, we performed molecular dynamics simulations of the SARS-CoV-1 FP and Ca2+ions. The preferred residue pairs for Ca2+ binding we identified (E801/D802, E801/D830, and D812/E821) include the two residues found to be essential for S function in our biological studies (D802 and D830). The three preferred Ca2+ binding pairs were also predicted to promote FP membrane insertion. We also identified a Ca2+ binding pair (E821/D825) predicted to inhibit FP membrane insertion. We then carried out simulations in the presence of membranes and found that binding of Ca2+ to SARS-CoV-1 FP residue pairs E801/D802 and D812/E821 facilitates membrane insertion by enabling the peptide to adopt conformations that shield the negative charges of the FP to reduce repulsion by the membrane phospholipid headgroups. This calcium binding mode also optimally positions the hydrophobic LLF region of the FP for membrane penetration. Conversely, Ca2+ binding to the FP E801/D802 and D821/D825 pairs eliminates the negative charge screening and instead creates a repulsive negative charge that hinders membrane penetration of the LLF motif. These computational results, taken together with our biological studies, provide an improved and nuanced mechanistic understanding of the dymanics of SARS-CoV-1 calcium binding and their potential effects on host cell entry.

Antibodies targeting the fusion peptide on the HIV envelope provide protection to rhesus macaques against mucosal SHIV challenge.

The fusion peptide (FP) on the HIV-1 envelope (Env) trimer can be targeted by broadly neutralizing antibodies (bNAbs). Here, we evaluated the ability of a human FP-directed bNAb, VRC34.01, along with two vaccine-elicited anti-FP rhesus macaque mAbs, DFPH-a.15 and DF1W-a.01, to protect against simian-HIV (SHIV)BG505 challenge. VRC34.01 neutralized SHIVBG505 with a 50% inhibitory concentration (IC50) of 0.58 μg/ml, whereas DF1W-a.01 and DFPH-a.15 were 4- or 30-fold less potent, respectively. VRC34.01 was infused into four rhesus macaques at a dose of 10 mg/kg and four rhesus macaques at a dose of 2.5 mg/kg. The animals were intrarectally challenged 5 days later with SHIVBG505. In comparison with all 12 control animals that became infected, all four animals infused with VRC34.01 (10 mg/kg) and three out of four animals infused with VRC34.01 (2.5 mg/kg) remained uninfected. Because of the lower potency of DF1W-a.01 and DFPH-a.15 against SHIVBG505, we infused both Abs at a higher dose of 100 mg/kg into four rhesus macaques each, followed by SHIVBG505 challenge 5 days later. Three of four animals that received DF1W-a.01 were protected against infection, whereas all animals that received DFPH-a.15 were protected. Overall, the protective serum neutralization titers observed in these animals were similar to what has been observed for other bNAbs in similar SHIV infection models and in human clinical trials. In conclusion, FP-directed mAbs can thus provide dose-dependent in vivo protection against mucosal SHIV challenges, supporting the development of prophylactic vaccines targeting the HIV-1 Env FP.

Fusion peptide-directed antibodies: Humoral armor against HIV-1 infection.

Infused neutralizing antibodies to the fusion peptide of the HIV envelope glycoprotein protected macaques from mucosal viral challenge (Pegu et al.).

Functionalized 3D Hydroxyapatite Scaffold by Fusion Peptides-Mediated Small Extracellular Vesicles of Stem Cells for Bone Tissue Regeneration.

3D printing technology offers extensive applications in tissue engineering and regenerative medicine (TERM) because it can create a three-dimensional porous structure with acceptable porosity and fine mechanical qualities that can mimic natural bone. Hydroxyapatite (HA) is commonly used as a bone repair material due to its excellent biocompatibility and osteoconductivity. Small extracellular vesicles (sEVs) derived from bone marrow mesenchymal stem cells (BMSCs) can regulate bone metabolism and stimulate the osteogenic differentiation of stem cells. This study has designed a functionalized bone regeneration scaffold (3D H-P-sEVs) by combining the biological activity of BMSCs-sEVs and the 3D-HA scaffold to improve bone regeneration. The scaffold utilizes the targeting of fusion peptides to increase the loading efficiency of sEVs. The composition, structure, mechanical properties, and in vitro degradation performance of the 3D H-P-sEVs scaffolds were examined. The composite scaffold demonstrated good biocompatibility, substantially increased the expression of osteogenic-related genes and proteins, and had a satisfactory bone integration effect in the critical skull defect model of rats. In conclusion, the combination of EVs and 3D-HA scaffold via fusion peptide provides an innovative composite scaffold for bone regeneration and repair, improving osteogenic performance.

A strategy for producing isotopically labeled peptides with antimicrobial activity or with short in vivo lifetime in Escherichia coli

AbstractEngineered Escherichia coli (E. coli) strains have been widely used to produce isotopically labeled peptides for NMR characterization on their structures and interactions. However, production of antimicrobial peptides (AMPs) by E. coli is still challenging, because AMPs are toxic to E. coli host and would lead to cell death after induction. On the other hand, expression of short peptides in E. coli host often encounter problems of the short in vivo lifetime of the peptides, which were rapidly degraded by endogenous enzymes during expression and purification steps. This report presents a practical method for overcoming these bottlenecks to enable E. coli to express AMPs and peptides that have short in vivo lifetime. This design uses the fusion of thioredoxin tags at both the N‐ and C‐termini of the target peptides. The steric effect of the large soluble tags at both ends of the peptide reduces peptide accessibility, thereby enhancing their in vivo stability and eliminating the toxicity associated with AMPs. The approach was validated using an AMP A3K/L7K‐LAH4 (K3K7) and a membrane fusion peptide (FP), which is a segment of the spike protein of SARS‐CoV‐2 and functions in fusing viral membranes and host cell membranes. Fusion expression of K3K7 with a thioredoxin tag only at the N‐terminal resulted in high toxicity to the host cells, leading to impaired cell growth and a failure to obtain expressed fusion protein. In contrast, the fusion proteins from both termini were successfully expressed and purified. In the case of the FP, the fusion of thioredoxin at both termini significantly enhanced its stability, protecting it from enzymatic degradation during expression and purification steps. On the contrary, the FP with thioredoxin fused only at the N‐terminal was found to be unstable in E. coli host strains. As stable isotope labeling on peptide is essentially important in NMR‐based structure and interaction studies, we also demonstrated that the developed approach enables efficient 15N labeling for NMR studies. This strategy may also be extended to produce other challenging peptides.

Pleomorphic viruses establish stable relationship with marine hyperthermophilic archaea.

Non-lytic viruses with enveloped pleomorphic virions (family Pleolipoviridae) are ubiquitous in hypersaline environments across the globe and are associated with nearly all major lineages of halophilic archaea. However, their existence in other ecosystems remains largely unknown. Here, we show that evolutionarily-related viruses also infect hyperthermophilic archaea thriving in deep-sea hydrothermal vents. Archaeoglobus veneficus pleomorphic virus 1 (AvPV1), the first virus described for any member of the class Archaeoglobi, encodes a morphogenetic module typical of pleolipoviruses, including the characteristic VP4-like membrane fusion protein. We show that AvPV1 is a non-lytic virus chronically produced in liquid cultures without substantially affecting the growth dynamics of its host with a stable virus-to-host ratio of ~1. Mining of genomic and metagenomic databases revealed broad distribution of AvPV1-like viruses in geographically remote hydrothermal vents. Comparative genomics, coupled with phylogenetic analysis of VP4-like fusogens revealed deep divergence of pleomorphic viruses infecting halophilic, methanogenic, and hyperthermophilic archaea, signifying niche separation and coevolution of the corresponding virus-host pairs. Hence, we propose a new virus family, "Thalassapleoviridae," for classification of the marine hyperthermophilic virus AvPV1 and its relatives. Collectively, our results provide insights into the diversity and evolution of pleomorphic viruses beyond hypersaline environments.

The critical role of aromatic residues in the binding of the SARS-CoV-2 fusion peptide to phospholipid bilayer membranes.

Based on the SARS-CoV-2 fusion peptide (FP) structure determined from the NMR experiment, we created six FP models under different environmental conditions to explore the effects of salt and cholesterol on FP-membrane binding. The all-atom molecular dynamics (MD) simulation results indicated that ionic environments notably impact the FP structure as well as the stability of the helical elements within the peptide. Our findings highlighted the unpredictable influence of ions on the secondary structures and dynamics of the FP, emphasizing the complexity and sensitivity of the peptide's conformations to ionic conditions. When exploring the peptide's interaction with a cholesterol-free phospholipid bilayer membrane, we found that the helical elements of the FP remain stable irrespective of the salt type (Na+ or Ca2+). This result emphasizes the crucial role of phospholipid bilayer membranes in supporting the secondary structures of the FP. The MD simulation results showed that Ca2+ ions facilitated deeper membrane penetration than Na+ ions, highlighting the critical role of calcium ions in the FP-membrane binding. Our study indicates the essential role of the aromatic residues (such as Phe833 and Tyr837) in the FP-membrane binding process. Finally, we investigated the FP-membrane binding patterns in the presence of cholesterol. The MD simulation results demonstrated that the coupling of Ca2+ ions and cholesterol would also benefit the FP-membrane binding. Furthermore, our findings reveal that while the type of ion and cholesterol content exert varied and unpredictable influences on FP-membrane binding patterns, aromatic residues like tyrosine (Tyr) and phenylalanine (Phe) play an essential role in FP-membrane binding. In particular, deep mutational scanning (DMS) experiments have confirmed that mutating phenylalanine in the FP significantly decreases viral mutational fitness, emphasizing the pivotal role of phenylalanine residues in membrane fusion. This knowledge can aid in developing more effective therapeutic strategies targeting the viral fusion peptide and its key amino acids, ultimately contributing to developing treatments and vaccines against the virus.

Metal element-fusion peptide heterostructured nanocoatings endow polyetheretherketone implants with robust anti-bacterial activities and in vivo osseointegration.

Polyetheretherketone (PEEK), renowned for its exceptional mechanical properties and bio-stability, is considered a promising alternative to traditional metal-based implants. However, the inferior bactericidal activity and the limited angiogenic and osteogenic properties of PEEK remain the three major obstacles to osseointegration in vivo. To overcome these obstacles, in this work, a versatile heterostructured nanocoating was conceived and equipped on PEEK. This nanocoating was designed to endow PEEK with the ability of photo-activated pathogen disinfection, along with enhanced angiogenesis and osteogenesis, effectively addressing the triple-barrier challenge towards osseointegration. The crafted nanocoating, encompassing diverse nutritional metal elements (Fe3+, Mg2+, and Sr2+) and a fusion peptide adept at promoting angiogenesis and osteogenesis, was seamlessly decorated onto PEEK. The engineered implant exhibited an antibacterial activity of over 94% upon near-infrared illumination by virtue of the photothermal conversion of the polyphenol nanocoating. Simultaneously, the decorated hierarchical nanocoatings synergistically promoted cellular adhesion and proliferation and up-regulated angiogenesis-/osteogenesis-associated cytokine expression in endothelial/osteoblast cells, resulting in superior angiogenic differentiation and osteoinductive capability in vitro. Moreover, an in vivo assay in a rabbit femoral defect model revealed that the decorated implant can achieve ameliorative osseointegrative fixation. Collectively, this work offers a practical and instructive clinical strategy to address the triple-barrier challenge associated with PEEK-based implants.