Protein and Peptide Letters - Current Issue

Peak-shouldering elution behavior was a common and unexpected result in bind-and-elute mode Cation Exchange Chromatography (CEX), which may be due to the pH transition during the elution step and the aggregation tendency of target proteins.

Improving the concentration of acid-base pairs in the wash buffers or elution buffers without changing pH or conductivity effectively resolved the peak-shouldering issue in CEX.

In the case of molecule A, the shoulder peak was eliminated in the CEX run by increasing the NaAc-HAc concentration from 50 mM to 100 mM in the elution buffer or from 50 mM to 75 mM in the wash buffer. Higher NaAc-HAc concentrations affect the pH transition in the early stages of the elution step, which may explain the elimination of the shoulder peak. A similar result was observed for molecule B, where increasing the Tris-HCl concentration in the elution buffer from 50 mM to 80 mM also removed the shoulder peak during elution.

The successful elimination of peak-shouldering behavior by increasing acid-base pair concentrations highlights the critical role of buffer capacity in modulating pH transitions during CEX. While this strategy offers a simple and effective solution, further investigation is needed to assess its applicability across diverse protein types and buffer systems.

These results demonstrate that increasing the concentration of acid-base pairs in the elution buffer or wash buffer of CEX using NaAc-HAc or Tris-HCl buffers is an effective strategy for eliminating the shoulder-peak.

Endometrial carcinoma (EC) incidence and mortality continue to rise, and reliable therapeutic targets remain scarce. We aimed to define the oncogenic role and mechanism of tumor protein D52 (TPD52) in EC, focusing on epithelial–mesenchymal transition (EMT) and the PI3K/AKT and ERK/MAPK signaling pathways.

In this study, we assessed the expression levels of TPD52 in EC tissues and benign endometrial tissues using immunohistochemistry. To further investigate the role of TPD52, we performed experiments both in vitro and in vivo. We transfected siRNA and overexpression (OE) plasmids into Ishikawa and HEC-1-A cell lines to knock down (KD) or overexpress TPD52, respectively. We observed the effects of TPD52 knockdown on tumor growth and EMT through in vitro experiments.

TPD52 was significantly upregulated in EC tissues compared with those of benign endometrial tissues. Silencing TPD52 significantly inhibited cell proliferation, migration, and invasion, whereas TPD52 overexpression produced the opposite effects. TPD52 facilitates epithelial-mesenchymal transition (EMT). Moreover, TPD52 stimulates the PI3K/AKT and ERK/MAPK signaling pathways.

These data position TPD52 as a bona fide EC oncoprotein that drives EMT via dual PI3K/AKT–ERK/MAPK signaling. Limitations include the modest patient cohort and the lack of clinical–pathological correlation analyses.

TPD52 promotes EC progression through EMT and PI3K/AKT and ERK/MAPK activation, offering a promising therapeutic target whose clinical utility warrants further investigation.

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.

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

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.

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.

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

Liver cancer is the third leading cause of cancer-related death. Plant-derived 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.

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.

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.

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.

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).

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.

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.

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.

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.