Protein and Peptide Letters最新文献

Introduction: Ubiquitin and the Ubls family are known for their high solubility and excellent expression profiles in recombinant systems. In contrast, Neural Precursor Cell Expressed, Developmentally Down-Regulated 8 (NEDD8) is a ubiquitin-like modifier that shares more than 60% sequence identity with ubiquitin and exhibits a similar structural fold. NEDD8 primarily functions by modifying the cullin subunits of cullin-RING E3 ligases, thereby playing a critical role in regulating the cell cycle, embryonic development, and DNA repair processes, particularly by localizing to sites of DNA damage. Despite its structural and functional similarity to highly soluble ubiquitin family proteins, recombinant NEDD8 is predominantly expressed in inclusion bodies, making its purification challenging.

Methods: Traditional refolding and purification strategies using 6M urea have proven inefficient in recovering properly folded and functional protein. In this study, we present a streamlined, high- -yield method for purifying NEDD8 based on on-column refolding using a 6xHis tag in combination with nickel-affinity chromatography, followed by size exclusion chromatography for further purification.

Results: The structural integrity and correct folding of the purified NEDD8 were confirmed through both nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy, validating the effectiveness of the method for producing biologically relevant, properly folded protein. Limitation of the Study: We applied an on-column refolding method for NEDD8, eliminating dialysis- associated losses and yielding well-folded protein. The approach is effective for small proteins but limited by size, hydrophobicity, and charge-related aggregation risks. Broader applicability requires case-specific optimization to ensure correct folding and structural fidelity across diverse proteins.

Conclusion: Our results demonstrate that this on-column refolding approach significantly improves the yield and refolding efficiency of NEDD8 compared to previous urea-based methods.

Introduction: Prolyl 4-hydroxylase beta peptide (P4HB) is a novel diagnostic and prognostic marker associated with cancer progression and clinical outcomes, and it is upregulated in multiple types of cancer cells. However, the influence and potential mechanisms of P4HB on the migration, invasion, and epithelial-mesenchymal transition (EMT) of bladder cancer cells remain unclear. This study aims to clarify the role of P4HB in the migration, invasion, and EMT of bladder cancer cells and to explore its potential mechanism related to the Claudin-1/AMPK/TGF-β1 pathway.

Methods: The mRNA and protein expression levels of P4HB were examined in human ureteral epithelial cells (SV-HUC-1) and five bladder cancer cell lines (J82, T24, 5637, UM-UC-3, and RT4). Stable cell lines with P4HB overexpression and knockdown were constructed, and the effects of P4HB on migration, invasion, EMT, and the expression of EMT-related genes in bladder cancer cells were analyzed using wound healing assays, Transwell invasion assays, cellular morphology observations, real-time quantitative PCR, in-cell western blotting, western blotting, and enzymelinked immunosorbent assays. Furthermore, Claudin-1 siRNA was transfected into P4HBoverexpressing cells to investigate its potential role in P4HB-induced invasion and EMT in bladder cancer cells.

Results: P4HB mRNA and protein expressions were significantly upregulated in human bladder cancer cell lines compared to those in ureteral epithelial cells. Cell migration, invasion, and EMT were significantly promoted in P4HB-overexpressing stable bladder cancer cells and suppressed in P4HB-knockdown cells. Furthermore, interference with P4HB downregulated EMT-related Claudin-1 mRNA and protein expressions and regulated the expression of downstream genes and proteins of Claudin-1. Moreover, interference of Claudin-1 with its siRNA significantly reversed the invasion and EMT induced by P4HB-overexpression, however, the effect of Claudin-1 siRNA was revised by TGF-β1 agonist and AMPK inhibitor.

Conclusion: P4HB promoted migration, invasion, and EMT of bladder cancer cells by activating the Claudin-1/AMPK/TGF-β1-related pathway.

Background: Adenine phosphoribosyltransferase (APRT) is an enzyme that facilitates adenosine monophosphate (AMP) biosynthesis by transferring a phosphoribosyl group to adenine using phosphoribosyl pyrophosphate as a donor. While the human enzyme is well characterized, structural insights into bacterial APRTs remain limited. Fusobacterium nucleatum is associated with periodontal disease, yet its APRT enzyme (FnAPRT) has not been structurally investigated.

Objective: This study aimed to examine the crystal structure of FnAPRT and ligand-induced conformational changes to understand its enzymatic and substrate recognition mechanisms.

Methods: The FnAPRT protein was heterologously expressed in Escherichia coli, followed by initial purification using nickel-charged affinity resin chromatography and further purification through size-exclusion chromatography. The FnAPRT structure was resolved using X-ray crystallography and compared with that of E. coli APRT (EcAPRT), exhibiting the highest amino acid sequence similarity among bacterial APRT structures.

Results: AMP and phosphate (PO4) were observed in the active site of FnAPRT. Significant differences in ligand positioning were observed between the AMP-PO4-bound structures of FnAPRT and EcAPRT. Structural shifts induced by AMP-PO4 binding were detected. The Arg78 and Lys82 residues from the alternate subunit occupied the PO4 site in the absence of ligands, but they interacted with PO4 upon AMP-PO4 binding. Structural comparison of the AMP-PO₄-bound FnAPRT with that of the adenine-bound EcAPRT highlighted variations in the adenine-binding site and associated structural changes.

Discussion: Structural comparison of the AMP-PO4-bound FnAPRT with that of the adeninebound EcAPRT highlighted variations in the adenine-binding site and the associated structural changes.

Conclusion: The AMP-PO₄-bound FnAPRT exhibited distinct ligand-binding modes despite sharing a high sequence similarity with EcAPRT. The structures demonstrated ligand movement during bacterial APRT reactions.

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.

Scorpion venom compounds are known to contain nucleotides, polypeptides, mucoproteins, lipids, biogenic amines, and other unidentified macromolecules. Several peptides in scorpion fluids have demonstrated a wide range of biological activities with strong specificity for their targeted sites. Margatoxin, isolated from the venom of the scorpion, exhibits desirable properties, including high selectivity, good permeability, and stability in cancer cells, which can be achieved at picomolar doses, thereby blocking voltage-gated K+ channels. This narrative review consolidates results from an extensive literature search conducted in major electronic databases up to September 2024. Important studies were identified using keywords associated with scorpion venom peptides, Kv1.3 channels, cancer treatment, and neurodegenerative disorders. The amino acids that make up Margatoxin have an effective molecular function in blocking voltage-gated K+ channels 1.3. Due to the abnormally high expression of voltage-gated K⁷ channel 1.3 in various types of cancers, blockers of this channel can inhibit apoptosis, metabolic changes, tumor angiogenesis, invasion, and migration. On the other hand, these channel blockers have emerged as a promising therapeutic approach for neurological disorders, such as Alzheimer's and Parkinson's diseases. The strong efficacy and targeted action of margatoxin further position it as a promising drug candidate. As the number of individuals affected by cancer and neurological conditions continues to rise, research into scorpion venom peptides like margatoxin may lead to innovative therapeutic options for future treatments.

Holoproteomics is a state-of-the-art advancement in spatial proteomics, enabling comprehensive spatial analysis of proteins in tissue microenvironments by combining Digital Holographic Microscopy (DHM) imaging and high-precision laser capture microdissection (LCM) techniques. DHM is an advanced technology that utilizes optical interference principles for non-invasive imaging, providing high-resolution three-dimensional visualization of cells and macromolecules without requiring markers. In proteomics, DHM provides crucial technical support for investigating protein interactions and enables high-precision tracking and analysis of dynamic protein changes. In this review, we systematically survey peer-reviewed literature published in the past five years, with a focus on experimental and clinical studies applying DHM to proteomic analyses. Based on the significant advantages of this technology, we introduce the concept of "Holographic proteomics" as an emerging research field with promising future directions.

Background: The use of peptides in the pharmaceutical and cosmetic industries is attracting increasing attention. Most of the peptides currently marketed are obtained by chemical processes, most frequently solid-phase peptide synthesis (SPPS).

Objective: Although SPPS is efficient, it requires hazardous solvents, such as N,Ndimethylformamide, dichloromethane, and N-methylpyrrolidone, as well as the bases piperidine and 4-methylpiperidine in the deprotection step. This study presents two alternative reagents, 2- aminoethanol and 2-amino-2-methyl-1-propanol, for the removal of the fluorenylmethyloxycarbonyl protecting group used in SPPS.

Methods: The traditional and alternative green SPPS using Fmoc protocol were employed.

Results: The use of these reagents in SPPS afforded two peptides in high yield in an environmentally sustainable solvent.

Conclusion: The reagents are thus promising alternatives to piperidine derivatives, particularly 2- amino-2-methyl-1-propanol, in SPPS.

Background: Aloperine (ALO) is a vital alkaloid present in the traditional Chinese herb Sophora alopecuroides, which has demonstrated effective anti-inflammatory activity. However, the effects and the mechanism of action of ALO on cisplatin (CDDP)-induced nephrotoxicity remain unclear.

Objective: This study aimed to investigate the effects of ALO on CDDP-induced nephrotoxicity and its potential mechanism of action in vitro.

Methods: Cell viability, lactate dehydrogenase cytotoxicity, apoptosis, activity of Caspase-Glo 3/7 and 1, in-cell western blotting, immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA) were performed to assess the influence of ALO on CDDP-treated kidney cells. Inhibitors of phosphatidylinositol 3-kinase (PI3K, LY294002), protein kinase B (Akt, AKT inhibitor VIII), and nuclear factor kappa B (NFκB, BAY 11-7082) were used to determine their potential mechanisms of action.

Results: The results indicated that ALO significantly reversed the inhibition of cell viability, cytotoxicity, apoptosis, and the release of inflammatory factors induced by CDDP in kidney cells. ALO attenuated the PI3K/AKT/NFκB-mediated pathway activated by CDDP treatment and downregulated the CDDP-induced nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome. Furthermore, the PI3K and AKT inhibitors diminished the effects of ALO on CDDP-treated kidney cells. Additionally, NFκB inhibitors reversed the effects of the PI3K and AKT inhibitors on ALO in CDDP-treated kidney cells.

Conclusion: These results suggest that ALO protects against CDDP-induced injury in kidney cells by modulating the PI3K/AKT/NFκB-mediated NLRP3 inflammasome.

Introduction: Liver cancer is the third leading cause of cancer-related death. Plantderived therapeutics have played a significant role in preventing and treating many diseases, including cancers. The present study investigated the anticancer properties of protein fractions from the green leaf extract of Adenium obesum (A. obesum) in the laboratory.

Methods: Protein fractions of leaf extract were separated using reversed-phase high-performance liquid chromatography (RP-HPLC). The cytotoxicity of protein fractions was studied by MTT and sulforhodamine B assays. The apoptotic cell death was examined using the alkaline comet assay, and redox-related indicators were assessed using the catalase enzyme activity assay, glutathione content, and nitric oxide release. The RBC hemagglutination test investigated the possible presence of ribosome-inactivating proteins (RIPs) in the most toxic protein fraction, and the LD50 of the protein fraction with the highest anticancer effects was determined. The amino acid sequence of fraction proteins was determined by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method.

Results: The results showed that protein fraction 8 had the highest toxicity in the HepG2 cell line, with an IC50 of 0.16 μg/mL. This fraction induced hemagglutination in red blood cells at concentrations higher than 65 μg/mL. The apoptosis was induced in the HepG2 cells following treatment with the concentrations of 0.08, 0.16, 0.32, and 0.64 μg/mL. Moreover, the redox potential of the treated cells was changed after treatment. The in vivo cytotoxicity investigation of this fraction in mice showed that it is not toxic for animals in concentrations up to 800 μg/kg, indicating its safety potential for pharmaceutical applications. The protein extract in the aforementioned fraction contained two proteins (22 and 53 kD) as determined by electrophoresis and sequencing methods.

Conclusion: The findings of this investigation demonstrated that the protein content of fraction 8 derived from A. obesum leaf extract possesses anticancer activity in the HepG2 cell line. The two isolated proteins from this fraction are novel and have been reported for the first time. Further investigations should be performed to evaluate the treatment potential in in vitro/vivo conditions.