Protein and Peptide Letters - Volume 32, Issue 5, 2025

Canonical Wnt (Wnt/β-catenin) signaling maintains bone homeostasis by promoting osteoblastic activities. The inhibitory factor, Dickkopf-1 (DKK1), enhances bone resorption in malignant diseases. Low-density lipoprotein-related protein (LRP) 5 is antagonized by DKK1. This study aimed to investigate the expression of DKK1 and LRP5 in renal cell carcinoma bone metastasis (RCC-BM).

RCC-BM patients with paired samples of primary and metastatic lesions were selected for the study (RCC-BM group). RCC patients without any metastasis served as the control group (RCC-only group). Immunohistochemical staining with monoclonal anti-DKK1 and polyclonal anti-LRP5 antibody was conducted on paraffin-embedded slides. The staining results were recorded using scoring according to staining intensity in the renal tissue adjacent to the tumor, primary RCC lesions, and RCC-BM lesions.

DKK1 was differently expressed among normal renal tissues, primary RCC, and RCC-BM tissues (p<0.001). The DKK1 expression in primary RCC was significantly lower than that in normal renal tissues (p<0.001) without a difference between the RCC-BM and RCC-only groups. DKK1 expression in bone metastasis was significantly higher than that in primary tumors (p<0.001). For RCC-BM patients, the expression of LRP5 in the primary tumor was significantly lower than that in adjacent renal tissues (p<0.01). The tendency of lower expression was found in primary RCC from RCC-BM patients compared to RCC without metastasis (p=0.073).

A “rebound” pattern of DKK1 expression in bone metastasis lesions and the decreasing LRP5 expression in primary lesions of RCC-BM patients suggested that Wnt/β-catenin signaling was inhibited in RCC-BM. The overexpression of DKK1 and reduced expression of LRP5 suggest that these markers may be useful for the early prediction of RCC-BM.

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly pathogenic human coronavirus (CoV). For the treatment of COVID-19, various drugs, ayurvedic formulations, used for other diseases, were repurposed. Ayurveda and yoga exhibited a pivotal role in the treatment of COVID-19. Various medicinal plants, including garlic, tulsi, clove, cinnamon, ginger, black pepper, and turmeric, are recommended for the prevention of COVID-19 as immunity boosters along with their antiviral property.

In view of the drug repurposing approach, the present work has been initiated with the broader objectives of screening and identification of phytoconstituents of Indian spices against targets, namely furin, 3C-like protease (3CL-PRO), NSP-9 RNA binding protein, papain-like protease, RNA dependent RNA polymerase (RDRP), spike protein concerned with life cycle of SARS-CoV-2 using in-silico tools.

The phytoconstituents of Indian spices were screened for interaction with several targets using a molecular docking approach with the help of Discovery Studio 4.5 software. Furthermore, the pharmacokinetic analyses of selected ligands using ADMET and Lipinski’s rule of five were also performed.

In the present study, a total of 37 active phytoconstituents of Indian spices were screened for interaction with several identified targets of COVID-19 using a molecular docking approach. The ligands, namely morin, gingerol, myristic acid, quercetin, gallic acid and alliin were found to be the top interacting ligands with the targets analyzed.

Based on the present in-silico finding, the active components of spices could be considered for drug-lead compounds against COVID-19.

Gene fusion techniques have yielded promising results in the fusion of thermostable polymerases (Taq and Pfu) with single-stranded and double-stranded DNA-binding proteins. Constructing a terminal deoxynucleotidyl transferase (TdT) fusion enzyme with DNA-binding protein domains can enhance thermostability and broaden the enzyme's application field. This makes it a promising candidate for cost-effective de novo DNA synthesis and a more effective tool for demonstrating apoptosis and detecting viral DNA/RNA.

The design of fusion proteins was based on molecular dynamics and homology modeling. Native and fusion proteins were isolated using affinity chromatography on HisTrap HP. Thermostability was assessed through differential scanning fluorimetry and dynamic light scattering. HPLC analysis was conducted to evaluate enzyme activity.

According to the in silico predictions of the fusion protein structure, a homotetramer was formed. The expressed fusion proteins were successfully purified under native conditions, similar to TdT. The total yields of the studied proteins were 130 mg/L for single-stranded binding protein from E. coli (EcSSB), 5 mg/L for TdT, 9 mg/L for TdT_L1_EcSSB, and 7 mg/L for TdT_L2_EcSSB. The measured radius of TdT (3.5 nm) was found to be consistent with a monomeric structure; however, the fusion proteins were expected to form a homotetramer. Additionally, fusion with EcSSB was found to prevent aggregation, which positively affected the thermal stability of the fusion protein. Instead of elongating the substrate by adding nucleotides, the fusion enzyme removed a nucleotide, specifically TTP, from the 3'-end of the DNA strand.

The fusion of TdT with EcSSB resulted in increased thermal stability and a reduced ability to add nucleotides to the substrate.

Molting and reproduction play vital roles in the life cycle of brachyuran crabs, and these two processes are closely interconnected. A key player in the molting cycle is cryptocyanin, which is similar to hemocyanin in sequence, size, and structure. Hemocyanin is a copper-containing oxygen-binding protein, while cryptocyanin is a copper-free protein that lacks oxygen-binding capacity.

The goal of the study was to carry out the isolation, cloning, and expression of the partial cryptocyanin gene from the Indian variety of Scylla olivacea.

The partial cryptocyanin gene was isolated from the hemocytes of the S. olivacea male and female crabs by qPCR for comparative expression analysis of the cryptocyanin gene.

We successfully amplified, cloned, and expressed a 519 bp partial cDNA encoding cryptocyanin from the Indian variety of Scylla olivacea, within the pRSET-B vector.

In this study, we conducted a comprehensive analysis of cryptocyanin expression in male and female crabs during the intermolt stage. Our findings revealed Cq values of 28.97 for males and 33.68 for females, highlighting a significantly lower abundance of cryptocyanin protein in female crabs.

Our study showed that crustacean cDNA can be effectively expressed in bacterial vectors, and clones were stable for up to 6 months at -80°C. Real-time data showed a significant difference in cryptocyanin levels between male and female crabs. This finding highlights the need for further research with a larger sample size for better understanding.

Human papillomavirus type 16 (HPV16) is implicated in various malignancies. The virus enters host cells through endocytosis, during which the minor capsid protein L2 interacts with the S100A10 subunit of the annexin A2 heterotetramer (A2t) on the host cell membrane. This interaction is critical for facilitating HPV entry and subsequent infection of human cells. Therefore, examining the interaction between the L2 protein and S100A10 is crucial for advancing our understanding of the mechanisms by which HPV16 infiltrates cells.

The cell-free expression (CFE) system was investigated for L2 purification. The structure of L2 was characterized and its interaction with S100A10 was explored.

The L2 protein was expressed using a CFE expression system, and its expression was verified via Western blotting. L2 was further purified through size-exclusion chromatography (SEC), and its structural features were preliminarily assessed using transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Additionally, surface plasmon resonance (SPR) was employed to analyze the interaction between L2 and S100A10.

Western blotting confirmed the successful expression of L2. TEM and CD provided preliminary structural observations of L2, and SPR measurements yielded precise kinetic parameters for the interaction between L2 and S100A10.

In this study, we successfully expressed the HPV16 L2 protein using a cell-free protein expression system. Preliminary structural analysis using TEM and CD revealed key structural features of L2. Furthermore, SPR analysis provided detailed kinetic parameters for its interaction with S100A10. These findings provide more details on understanding L2’s structural features, with broader implications for antipathogen studies.