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Protein and Peptide Letters - Online First
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Molecular Cloning and Expression of Cryptocyanin Gene Isolated from an Indian Variety of Scylla olivacea
Authors: Simran Mann, Chittibabu Shanthi and Manu AsthanaAvailable online: 21 May 2025More LessBackgroundMolting 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.
ObjectiveThe 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.
MethodsThe 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.
ResultsWe successfully amplified, cloned, and expressed a 519bp partial cDNA encoding cryptocyanin from the Indian variety of Scylla olivacea, within the pRSET-B vector.
ConclusionOur study showed that crustacean cDNA can be effectively expressed in bacterial vectors, and clones were stable for up to 6 months at -80oC. 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.
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Inhibition of Canonical Wnt Signaling in Renal Cell Carcinoma Bone Metastasis: An Immunohistochemical Analysis of DKK1 and LRP5 Expression
Authors: Zixiong Huang, Yiqing Du, Huaqi Yin, Gongwei Wang and Tao XuAvailable online: 07 May 2025More LessBackground and ObjectiveCanonical 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).
MethodsRCC-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.
ResultsDKK1 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).
ConclusionA “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.
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Unraveling the Role of Functional Amyloids and Amyloid Peptides in Disease Detection
Authors: Priyanka Kumar and Nandini SarkarAvailable online: 07 May 2025More LessAmyloid 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.
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Development of a Peptide-Based Multiepitope Vaccine from the SARS-CoV-2 Spike Protein for Targeted Immune Response Against COVID-19
Available online: 14 April 2025More LessBackgroundSince 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.
MethodsHere was employed immunoinformatics tools 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.
ResultsThe 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.
ConclusionOur 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.
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Use of Plant Peptidases for the Production of Therapeutic Peptides
Available online: 11 April 2025More LessPeptidases play crucial roles in numerous physiological processes within living organisms. Therefore, they have been employed in various pharmaceutical applications. Plant peptidases have attracted considerable attention in various areas due to their specificity, stability across a diverse range of pH and temperatures, and safety profile. Here, we have focused on the use of plant peptidases, mostly papain and bromelain, to produce biologically active peptides, which confer various health advantages, including antioxidant, antimicrobial, antihypertensive, analgesic, antidiabetic, and anti-inflammatory effects. We have also discussed the importance of the action mechanism of peptidases for generating bioactive peptides with specific sequences and functions, the ecological and sustainability benefits of plant-derived peptidases compared to animal alternatives, digestive stability and bioavailability of peptides, as well as some obstacles to the commercialization of bioactive peptides and key challenges in peptidase-based industrial applications. Finally, we have examined enzyme immobilization as a viable method to enhance the production of bioactive peptides, offering numerous advantages in both research and industry contexts.
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Unveiling the Potential Role of Hesperetin and Emodin as a Combination Therapy to Inhibit the Pancreatic Cancer Progression against the C-Met Gene
Available online: 24 March 2025More LessBackgroundPancreatic adenocarcinoma (PAAD) is one of the most prevalent cancers, and it has high death rates. Only 10% of PAAD patients can survive until 5 years. Hence, the improvement of survival rate of the patients should be improved.
AimThe present study used a computational approach to identify novel biomarkers and potentially effective small drug-like molecules in PAAD.
ObjectiveThe objective of this study was to identify the Differentially Expressed Genes (DEGs) and survival rate affecting genes (SDEGs) to single out the specific gene responsible for pancreatic cancer and predict the efficacy of interactions with hesperetin and emodin. Further, another objective was to validate the predicted efficacies using an MTT assay.
MethodsThe GEPIA2 database was used to analyze the TCGA-PAAD dataset and identify DEGs and SDEGs. Venn identified the commonly scattered genes between the DEGs and SDEGs. Network Analyst v3.0, CytoScape v3.10.1, and cytoHubbawere used to construct protein-protein interactions (PPI) network and identifying hub genes which were described as target proteins. The Protein Data Bank (PDB) and PubChem were utilized to obtain the PDB structure of the target proteins and 13 phytocompounds in SDF format. Molecular docking studies were carried out and visualized by utilizing Autodock vina and Discovery Studio Visualizer v19.1.0.1828. The cytotoxicity was measured in the MiaPaCa-2 cell line after being treated with hesperetin and emodin.
ResultsA total of 9219 Differentially Expressed Genes (DEGs) from the TCGA-PAAD dataset were identified. Among them, 8740 and 479 genes were up and down-regulated with the statistical significance of P ≤ 0.05, respectively. Likely, 500 most survival rate affecting genes (SDEGs) in PAAD patients with a statistical significance of P ≤ 0.05 were identified. The common 137 genes were identified between these obtained DEGs and SDEGs. The survival heat map was delineated for the predicted 137 common genes. Ninety-six genes were identified as the most hazardous genes (highlighted in red). After that, the network was constructed by using PPI for the most hazardous 96 genes. From the constructed PPI network, the highly interacted top 10 genes were identified. The survival analysis was carried out to identify the most hazardous genes and revealed that all the identified genes significantly reduced the survival rate of the patients affected by PAAD. From that, high survival affecting 5 genes, such as CDK1, CENPE, NCAPG, KIF20A, and c-MET, were selected for further analysis. The molecular docking studies were carried out for the identified top 5 genes, with the 13 phytocompounds reviewed previously for anti-cancer activity. The molecular docking analysis revealed that the hesperetin (binding affinity (BA) = -8.0 kcal/mol; Root mean square deviation (RMSD) = 2.012 Å) and emodin (BA = -8.6 kcal/mol; RMSD = 1.605 Å) interacted well with the c-MET based on the number of hydrogen bonds and BA. Hence, the synergistic efficacy was validated in the cell line MiaPaCa-2 with the hesperetin, emodin, and hesperetin: emodin in combination and obtained the IC50 values of 171.3 µM, 72.94 µM, and 92.36 µM respectively.
ConclusionThe results stated that emodin significantly reduced the cell proliferation rate of the MiaPaCa-2 pancreatic cells, and no synergistic effects were observed in this context with hesperetin. However, emodin improved the hesperetin efficacy in pancreatic cells, indicating that structural modification through pharmacokinetics by coupling these two compounds may help to identify novel compounds to treat pancreatic cancer in the future. However, further pancreatic cell lines, such as Panc-1, BxPC-3, etc., and in vivo models that include CDX and PDX are needed to verify the combination effect of hespertin and emodin on pancreatic cells.
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Plant-derived Cyclotides in Immunomodulation and their Therapeutic Potential
Authors: Reema Mishra, Preeti Agarwal, Anshita Sharma, Meenal Mittal, Pooja Gulati and Aparajita MohantyAvailable online: 18 March 2025More LessThe incidences of immune-related disorders have drastically increased in recent years across the world population. Treatment and management of these diseases, especially autoimmune disorders, are complex and challenging. Available synthetic drugs are not completely effective and also pose serious side effects for the patients. Cyclotides are a class of plant-derived cyclic peptides (28-37 amino acids) with three conserved disulfide linkages establishing a cyclic cystine knot (CCK) motif that makes them very stable biomolecules. Their inherent stability, bioavailability and membrane-penetrating capabilities render them attractive potential pharmacological agents. Studies have demonstrated that cyclotides can either enhance or suppress immune responses, making them versatile candidates for treating various immune-related disorders. Of more than 1000 cyclotides discovered to date, only up to 15 native cyclotides (e.g. kalata B1, pase and caripe cyclotides) have been screened to demonstrate their immunomodulatory activity. Of special significance is the chemically synthesised lysine mutant of kalata B1 viz. [T20K], where preclinical studies have shown promise in the treatment of the autoimmune disorder, multiple sclerosis. In vivo studies in mice models have demonstrated that daily administration of 1mg/day of [T20K] led to a significant decrease in the level of cytokine secretion, lesser demyelination (<1%) and very low inflammatory index (<0.5), in the immunized mice. Moreover, when compared with other immunosuppressive drugs (azathioprine, prednisolone, and cyclosporine A) there was a notable drop in mortality and morbidity in mice administered with [T20K]. The cyclotides, kalata B1 and MCoTI-I have also been used as scaffolds to graft bioactive peptides with immunomodulatory activity. Subsequent in vitro and in vivo studies of these grafted cyclotides have demonstrated their therapeutic ability. Keeping in view the therapeutic potential of cyclotides as immunomodulatory peptides, the present review discusses its current research scenario and implications for the future in tackling immune-related disorders.
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Diminazene Aceturate (DIZE) Ameliorates Hypertension and Induces Anxiolytic- and Antidepressant-like Effects in TGR(mRen2)27
Available online: 24 February 2025More LessIntroductionDiminazene aceturate (DIZE) was described as an angiotensin-converting enzyme 2 (ACE2) activator. ACE2/Angiotensin-(1-7)/Mas receptor axis presents protective actions on cardiovascular diseases and plays an important modulatory role in the neurobiology of mood and anxiety disorders.
ObjectivesTo evaluate the effects of chronic intracerebroventricular (ICV) treatment with DIZE on blood pressure, anxiety- and depression-like behaviors in hypertensive transgenic (mRen2)27 rats (TGR).
MethodsMale TGR and Sprague-Dawley rats (10-12 weeks old) were subjected to chronic ICV infusion of DIZE (1.0 µg/h for 7 days). Blood pressure and heart rate were measured by tail plethysmography and anxiety- and depression-like behaviors were evaluated through elevated plus maze, marble burying and forced swim tests, respectively.
ResultsTreatment with DIZE induced a significant reduction in mean arterial pressure in both TGR and SD rats. A decrease in heart rate was only observed in the hypertensive animals. Additionally, treatment with DIZE attenuated the anxiety- and depression-like behaviors that were observed in TGR.
ConclusionDIZE has central anti-hypertensive, anxiolytic, and anti-depressive effects.
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Inconsistent Protein Stability Despite Pre-HECT Domain Helix: Unveiling Variability in HECT Ligases
Authors: Çağdaş Dağ, Cansu Deniz Tozkoparan Ceylan and Cemre Sare CansızAvailable online: 11 February 2025More LessIntroductionUbiquitin 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.
MethodSome 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.
ResultThe 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.
ConclusionThe 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.
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