Protein and Peptide Letters - Volume 32, Issue 11, 2025

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.

Traditional refolding and purification strategies using 6M urea have proven inefficientin 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. 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.

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.

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

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.

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

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.

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.

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

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.

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.

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 enzyme-linked immunosorbent assays. Furthermore, Claudin-1 siRNA was transfected into P4HB-overexpressing cells to investigate its potential role in P4HB-induced invasion and EMT in bladder cancer cells.

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.

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

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).

Although SPPS is efficient, it requires hazardous solvents, such as N,N-dimethylformamide, 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 fluorenyl-methyloxycarbonyl protecting group used in SPPS.

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

The synthesis of two peptides showed that the 2-aminoethanol and 2-amino-2-methyl-1-propanol are viable replacements for piperidine-derived reagents in peptide synthesis.

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

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