Protein and Peptide Letters最新文献

Autophagy is a self-eating cellular process in which the cell breaks down worn-out organelles, damaged/defective proteins, and toxins. Impaired autophagy is a significant factor in the development of various metabolic disorders, along with oxidative stress, inflammation, mitochondrial and endoplasmic reticulum dysfunction. These disorders pose a significant health and economic burden on the global human population, owing to their steadily rising prevalence. Therefore, modulating the expression of proteins involved in the autophagy-related pathways can be a promising avenue for curbing the development and progression of these disorders. Humanin (HN) is a 24-amino acid mitochondrial-derived peptide. It possesses anti-oxidant, anti-inflammatory, and pro-apoptotic properties. The analogs of HN can be generated by replacing specific amino acids in the polypeptide chain, thereby functionally modifying the peptide. Among these, humanin- glycine (HNG) is the most widely studied analog in both in vivo and in vitro disease models. It is far more potent than HN, with a potency that is 1000 times greater. To the best of our knowledge, this review is the first to discuss and examine the available evidence regarding the potential involvement of HN or its analogs in regulating autophagy pathways. The review primarily highlights that HN is an autophagy inducer, which can promote cell survival in the presence of metabolic and oxidative stress, particularly the HNG analog. Future research is imperative to comprehensively evaluate the effects of HN and its analogs on autophagy. Further investigations are needed to correlate its levels with various autophagic markers in different metabolic diseases, offering the potential for groundbreaking discoveries in understanding disease mechanisms and developing novel therapeutic strategies.

Background: The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies).

Objectives: To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed.

Methods: These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy.

Results: Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions.

Conclusion: This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

Introduction: The shepherin II peptide is characterized by a histidine/glycine-rich sequence. This study aimed to design, express recombinantly, and evaluate the antiviral activity of shepherin II against hepatitis A virus (HAV).

Methods: The shepherin II gene was reverse-translated, cloned into the pET-3a vector, and expressed in E. coli BL21 (DE3) pLysS cells induced with 2 mM IPTG. Purification was achieved via cation exchange chromatography, and intact mass analysis using mass spectrometry was carried out. Cytotoxicity on normal Vero cells and antiviral activity on HAV were evaluated.

Results: The mass spectrometry confirmed a primary peptide fragment with a molecular weight of 3,421.30 Da (100% relative abundance). SDS-PAGE verified peptide expression. Cytotoxicity tests on Vero cells showed a CC₅₀ of 219.26 ± 7.91 μg/ml. Antiviral assay revealed an EC₅₀ of 113.92 ± 4.58 μg/ml against HAV, resulting in a selectivity index (SI) of 1.92. This SI indicates limited selectivity compared to the reference drug amantadine, which exhibited an EC50 of 5.67 ± 0.71 μg/ml and an SI of 53.41.

Discussion: The recombinant expression of shepherin II was successfully achieved and confirmed by mass spectrometry and SDS-PAGE. The peptide showed measurable antiviral activity against HAV.

Conclusion: This study demonstrated the feasibility of recombinant shepherin II production and assessed its antiviral activity. However, the limited selectivity index of shepherin II remains a challenge that needs to be addressed through molecular modification or alternative delivery strategies to improve its clinical potential.

Messenger RNA (mRNA) has gained increasing attention as a valuable tool to cure various human diseases, particularly malignant tumors. Such growing interest has been triggered largely by the phenomenal clinical success of mRNA vaccines developed using lipid nanoparticle (LNP) technology against COVID-19. mRNA may be used to produce cancer immunotherapies in numerous different ways, including cancer vaccines to induce or enhance immunity to tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). mRNA can also be used to adoptively transfer T-cells for the expression of antigen receptors, such as chimeric antigen receptors (CARs), therapeutic antibodies, and immunomodulatory proteins to re-engineer the tumor microenvironment. However, the therapeutic potential of mRNA-based cancer immunotherapy is not fully utilized due to a few limitations, such as mRNA instability, production of immunogenicity, and a lack of efficient in-vivo delivery methods. This review provides an overview of the current advancements and future directions of mRNA-based cancer therapies, including various delivery routes and therapeutic platforms. It addresses the mechanistic basis of mRNA cancer vaccines, non-replicating and self-amplifying mRNA, as well as their clinical development, personalized vaccines, and applications of mRNA for encoding antigen receptors, antibodies, and immunomodulatory proteins. Moreover, the review addresses nanoparticle-based platforms, such as lipid nanoparticles (LNPs), polymeric nanoparticles, and peptide-based nanoparticles, all used to improve the therapeutic effectiveness of mRNA-based drugs by improving their targeted delivery to tissues. This review aims to provide insights into the use of state-of-the-art mRNA-based cancer immunotherapy.

Background: Since the Coronavirus Disease (COVID-19) became a pandemic in late 2019, vaccination remains the primary approach to combating the virus. Nevertheless, the emergence of new variants poses challenges to vaccine efficacy. This study aimed to identify targets within the SARS-CoV-2 spike (S) protein to detect T-cell responses to the five variants of concern from SARS-CoV-2: Alpha, Beta, Delta, Gamma, and Omicron.

Methods: Herein, immunoinformatics tools were employed to develop a peptide-based vaccine targeting the spike protein of SARS-CoV-2 and its major variants, including Alpha, Beta, Delta, Gamma, and Omicron. The peptides were screened for antigenicity, toxicity, allergenicity, and physicochemical properties to ensure their safety and efficacy.

Results: The potential T-cell epitopes with high immunogenicity and IFN-γ induction, are essential for a robust immune response by a comprehensive computational analysis. Population coverage analysis revealed significant coverage across diverse geographical regions, with significant efficacy in areas heavily impacted by the pandemic. Molecular docking simulations revealed strong interactions between the selected peptides and major histocompatibility complex class I (MHC-I) molecules, indicating their potential as vaccine candidates.

Conclusion: Our study provides a systematic approach to the rational design of a peptide-based vaccine against COVID-19, providing insights for further experimental validation and development of effective vaccines.

Proteins and peptides play a crucial role in biological functions and contemporary therapeutic approaches; however, their clinical effectiveness is frequently hindered by swift renal clearance and enzymatic degradation. Peptides possess structured amino acid sequences that facilitate targeted drug delivery and enhance patient adherence. In contrast, proteins demonstrate intricate stability behaviors affected by pH and environmental conditions, requiring careful formulation strategies. Addressing these challenges necessitates a comprehensive understanding of stability and regulatory requirements. Regulatory agencies, including the FDA, EMA, and PMDA, require comprehensive stability testing per guidelines such as ICH Q5C and ICH Q1A(R2). This ensures meticulous management of factors such as temperature control, formulation optimization, and aggregation mitigation. Stability enhancement requires the application of innovative techniques, including protein engineering, lyoprotection, and nanoparticle encapsulation, in conjunction with ongoing quality monitoring. Integrating scientific expertise with regulatory standards enables researchers and pharmaceutical manufacturers to develop safe, effective, and compliant protein and peptide therapeutics for various patient populations.

Background: Bacillus thuringiensis Cry toxins are well known for their insecticidal properties, primarily through the formation of ion-leakage pores via α4-α5 hairpins. His¹⁷⁸ in helix 4 of the Cry4Aa mosquito-active toxin has been suggested to play a crucial role in its biotoxicity.

Objective: This study aimed to investigate the functional importance of Cry4Aa-His¹⁷⁸ through experimental and computational analyses.

Methods: Ten His¹⁷⁸-substituted Cry4Aa mutants (H178D, H178E, H178K, H178R, H178G, H178F, H178Y, H178S, H178C, and H178Q) were generated via site-directed mutagenesis and expressed in Escherichia coli. Toxin solubility was assessed in carbonate buffer (pH 10.0), and biotoxicity was tested against Aedes aegypti larvae. Trypsin-treated toxins were evaluated using fluorescent dye-release assays. Ion channel formation was studied in planar lipid bilayers (PLBs), and structural analysis was performed via MD simulations and sequence alignments with known Cry toxins.

Results: All His¹⁷⁸-substituted mutants were overexpressed as 130-kDa protoxin inclusions at levels comparable to the wild-type (WT). Replacing His¹⁷⁸ with nonpolar or bulky polar residues reduced Cry4Aa biotoxicity to less than 10%, while substitutions with small, moderately polar, or negatively charged residues retained 50-85% activity, consistent with their in vitro solubility. Selected bioactive mutants, H178C and H178D, retained membrane-perturbing ability, like trypsin- activated WT, while the bioinactive H178Y mutant exhibited decreased membrane permeability. All tested mutants, including WT, induced cation-selective channels in PLBs with ~130-pS conductance. Sequence-structure analysis indicated that Cry4Aa-His¹⁷⁸ likely forms a hydrogen bond with His²¹⁷, a conserved His residue in helix 5.

Discussion: Specific physicochemical properties of residue 178 are critical for optimal larvicidal activity, making it a promising target for engineering more potent mosquito-control toxins.

Conclusion: His¹⁷⁸ in Cry4Aa-α4 potentially forms a stabilizing hydrogen bond with α5-His²¹⁷, which maintains the structural integrity of the α4-α5 hairpin. This structural stability is essential for efficient membrane insertion and optimal larvicidal activity.

Amyloid refers to a specific quaternary structure characterized by fibrillar arrangements of proteins or peptides forming cross β-sheet architectures. Initially associated with diseases like Alzheimer's, amyloid was seen predominantly as pathological. However, recent research has revealed that amyloid also plays functional roles across various biological systems, from bacteria to mammals. The cross β-sheet structure of amyloid enables the transformation of soluble proteins into insoluble fibrils, providing high stability and a robust prion-like copying mechanism. However, recent research has revealed that amyloid also plays functional roles in various biological systems, such as biofilm formation in bacteria, aiding melanin biosynthesis in humans, and supporting the formation of fungal hyphae. Understanding the dual nature of amyloid-a pathological and functional entity-offers insights into disease mechanisms and therapeutic strategies. Recognizing the distinction between pathological and functional amyloids is crucial for advancing diagnostics and treatments. This review highlights the importance of functional amyloids (FAs), particularly in disease detection, underscoring their significant biological roles and potential applications.

Introduction/objective: Protein phosphatases act as counterparts to protein kinases and are considered crucial for the homeostatic balance of cell signalling. In contrast to kinases, which can be categorized according to their substrate specificity, phosphatases are versatile and can detect substrates with much less distinction; hence, it is challenging to identify the physiological phosphatase-substrate pair. The Oca1 of Saccharomyces cerevisiae is a putative protein tyrosine phosphatase (PTP) and is required for cell cycle arrest in response to oxidative stress. The Oca1 mutants are sensitive to mTOR inhibitors, such as caffeine and rapamycin, and are involved in the regulation of TOR function. In an earlier research work, the enzyme exhibited no in vitro phosphatase activity and it was suggested that post-translational modifications or additional factors are necessary for it to be functional.

Methods: The modeling of Oca1 was performed to gain insight into the structural aspects. The full- length enzyme, as well as the enzyme without the N-terminal extension, was cloned, expressed, and purified to homogeneity. The structure, function, and stability of the purified enzyme were assessed using circular dichroism, fluorescence, and visible spectroscopy studies.

Results: The Oca1 was expressed and purified from Escherichia coli. The enzyme has been found to be functional, stable, and exist in an extended monomeric form, with a molecular mass of about 27 kDa. The enzyme without the extended N-terminal random coil has also been functional and slightly more stable than the full-length Oca1.

Conclusion: The purified functional enzyme may be used to gain insights into the biochemical aspects and its role in bioengineering.

Introduction: Ubiquitin and ubiquitin-like systems play crucial roles across a wide range of organisms, from simple to complex. Among the three enzyme-mediated post-translational modification (PTM) steps, the ligation step is the most critical. HERC5, a member of the HECT ligase family, is one of the three enzymes involved in the ISGylation system. However, the precise start points and lengths of the HECT domains in HECT ligases are still under debate.

Methods: Some studies suggest the inclusion of an additional N-terminal alpha helix region within the HECT domain. To investigate the structural biology of the HECT domain of HERC5, we produced and purified various lengths of the HERC5 HECT domain using different fusion proteins. This approach allowed us to explore the role of the N-terminal alpha helix in the stability of the HECT domain. Our experiments successfully produced and purified HERC5 HECT domains of different lengths with various fusion proteins.

Results: The findings demonstrated that the N-terminal alpha-helix does not enhance the stability of the HECT domain. These results challenge the notion that the N-terminal alpha-helix should be generally included in the HECT domain across all HECT ligases.

Conclusion: The inclusion of this region within the HECT domain may not be appropriate for generalization, as it does not contribute to stability, contrary to some previous suggestions.