Protein and Peptide Letters最新文献

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.

Background: Natural killer (NK) cells, as part of the group I innate lymphocytes (ILCs) are essential for tumor immune surveillance. NK cells can recognize and eliminate target cells without the need for prior sensitization or restriction of major histocompatibility complexes (MHCs) and antigens. However, the limited infiltration of metastatic NK cells poses significant challenges for advancing adoptive cell immunotherapy for solid tumors.

Objective: This study aimed to explore the potential of using tumor penetrating peptide (TPP) iRGD to promote the delivery of activated NK cells to deeper layers of tumor tissue.

Methods: Flow cytometry was performed to evaluate the activation, inhibition, and expression of other receptors involved in cytotoxicity. High-pressure liquid chromatography (HPLC) and mass spectrometry were used to detect the purity of iRGD. 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-poly(ethylene glycol)-iRGD (DSPE-PEG-iRGD) was synthesized. Surface modification of cells was performed using DSPE-PEG-iRGD. Multicellular tumor spheroids (MCTSs) were established to evaluate permeability. In addition, in order to better simulate the physiological characteristics of solid tumors in vivo, we generated 3D spheroids from HGC27 gastric cancer cell line and BXPC-3 pancreatic cancer cell line to study the anti-tumor effect of NK cells with combination iRGD in vitro. The mouse models of gastric cancer and pancreatic cancer were used. In addition, the synergistic anti-tumor effects were evaluated in vivo based on the tumor volume and body weight of mice.

Results: Initially, we treated NK cells with interleukin-2 (IL-2), resulting in significant activation as indicated by upregulation of CD56. On the 15th day, the proliferation of CD3-/56+cell population in NK cell culture containing IL-2 significantly increased, and the NK cell amplification factor was greater than 300. In addition, NK cells exhibited increased cytotoxicity towards cancer cell lines. When the ratio of effect to target was 10:1, the killing rate of NK cells against BXPC-3 was 83.1%. iRGD modification enabled NK cells to penetrate MCTSs, resulting in cytotoxicity against target HGC27 and BXPC-3 cells. In addition, NK cells modified with iRGD significantly reduced tumor growth in the xenotransplantation model of gastric cancer and pancreatic cancer mice model.

Conclusion: In summary, our results indicated that NK cells exhibited higher efficacy and lifespan against cancer cell lines in vitro. Furthermore, the integration of iRGD into NK cells led to improved infiltration and targeted elimination of MCTSs. Moreover, the application of iRGDmodified NK cells has shown significant anti-tumor efficacy against solid tumors in vivo. This joint strategy may significantly improve the efficacy of NK cell immunotherapy in treating various solid tumors.

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.

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.

Members of the 14-3-3 protein family are involved in various cellular processes, including migration, angiogenesis, cell cycle, apoptosis, and signal transduction. Nevertheless, the 14-3-3 family possibly plays a fundamental role in the development of diseases and cancer by regulating various biological pathways. MicroRNAs (miRNAs) are mainly transcribed by RNA polymerase II (pol II), with only a few exceptions involving RNA polymerase III (pol III). They can control cell mechanisms through different pathways. miRNAs inhibit or destroy mRNAs by binding to them. They control intracellular mechanisms by binding to molecules such as the 14-3-3ζ protein. miRNAs play a role in regulating this protein, and by inducing or suppressing it, they contribute to either the development or the prevention of the diseases. Therefore, considering the importance of the 14-3-3ζ protein in different pathways within the body, we decided to investigate the relationship between miRNAs and 14-3-3ζ and clarify their interactions, in this review.

Introduction: The etiology of acute Achilles tendon rupture (ATR) remains unclear. This study conducted a comprehensive case-control study of the proteome profile to gain insights into the potential pathogenesis of acute ATR and identify novel biomarkers.

Methods: Serum (iTRAQ) and urine (label-free proteomics) from 15 acute ATR patients and 15 healthy controls were analyzed. Significant differential expression was defined as ≥1.2-fold (serum) or ≥2-fold (urine) change with p < 0.05. Bioinformatics analyses (GO, KEGG, PPI) were performed.

Results: 44 serum and 198 urine proteins were differentially expressed. Enriched pathways included immune response, metabolism, immune response, and redox regulation. protein-protein interaction analysis of the differentially expressed proteins (P < 0.05) highlighted abnormalities in major protein-protein interaction hubs, specifically pyruvate kinase (PKM), peroxiredoxin-1 (PRDX1), phosphoglycerate kinase 1 (PKG1), profilin-1, and apolipoprotein A-IV, observed in the serum and urine samples of acute ATR patients.

Discussion: Metabolic dysregulation may affect tendon structure/strength; redox imbalance could promote degeneration. Immune-related proteins may reflect injury responses. Glycolytic enzymes (PKM, PGK1) suggest disrupted energy metabolism.

Conclusion: Proteomic abnormalities in metabolism, immune, and redox pathways, along with key proteins (PKM, PRDX1, PGK1), may contribute to ATR pathogenesis, offering potential biomarkers warranting further validation.

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.

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: Cholelithiasis is the most prevalent inflammatory condition of the gallbladder. The regulation of biological processes, including energy homeostasis, and control of body weight are key mechanisms that the leptin and melanocortin pathways play a role in Cholelithiasis is the most prevalent inflammatory condition of the gallbladder. There are various risk factors for the development of gallstone disease, especially weight gain, and obesity is just one of them. This risk factor can be minimized by maintaining appetite and energy balance. Here, leptin and melanocortin pathways are the key mechanisms in maintaining appetite and energy homeostasis.

Objectives: The aim of this study was to investigate the relationship between the levels of LEP, LEPR, TrkB, BDNF, POMC, and MC4R proteins in patients with Cholelithiasis. This study aims to determine the relationship between LEP, LEPR, TrkB, BDNF, POMC, and MC4R protein levels, which play a role in maintaining appetite and energy homeostasis, and cholelithiasis.

Methods: This study examined 44 patients diagnosed with Cholelithiasis and 44 healthy control subjects who had not previously been diagnosed with any form of Cholelithiasis. The levels of leptin (LEP), Leptin Binds To Leptin Receptors (LEPR), Tropomyosin Receptor Kinase B (TrkB), Brain-Derived Neurotrophic Factor (BDNF), Pro-OpioMelanoCortin (POMC), and Melanocortin- 4 Receptors (MC4R) molecules were analyzed using the Enzyme-Linked Immunosorbent Assay (ELISA) method. The results were analyzed using the SPSS Software (Version 22.0) program and GraphPad Prism 8.0.1 software.

Results: The study found a statistically significant decrease (p < 0.05) in MC4R, TrkB, BDNF, and POMC protein levels in Cholelithiasis patients compared to the control group. There was no statistically significant difference in LEP and LEPR concentration values between the two groups (p = 0.247, p = 0.674).

Conclusion: The proteins MC4R, TrkB, BDNF, and POMC, which are involved in the leptin and melanocortin pathways may play a significant role in Cholelithiasis disease. However, more detailed research on the relevant proteins is needed. Nevertheless, this research will guide new studies.