Protein and Peptide Letters - Volume 30, Issue 9, 2023

Papillary thyroid carcinoma (PTC) is a common endocrine malignant tumor. The incidence of PTC has increased in the past decades and presents a younger trend. Accumulating evidence indicates that circular RNAs (circRNAs), featured with non-linear, closed-loop structures, play pivotal roles in tumorigenesis and regulate cell biological processes, such as proliferation, migration, and invasion, by acting as microRNA (miRNA) sponges. Additionally, due to their unique stability, circRNAs hold promising potential as diagnostic biomarkers and effective therapeutic targets for PTC treatment. In this review, we systematically arrange the expression level of circRNAs, related clinical characteristics, circRNA-miRNA-mRNA regulatory network, and molecular mechanisms. Furthermore, related signaling pathways and their potential ability of diagnostic biomarkers and therapeutic targets are discussed, which might provide a new strategy for PTC diagnosis, monitoring, and prognosis.

Background: The immune system is able to recognize substances that originate from inside or outside the body and are potentially harmful. Foreign substances that bind to immune system components exhibit antigenicity and are defined as antigens. The antigens exhibiting immunogenicity can induce innate or adaptive immune responses and give rise to humoral or cell-mediated immunity. The antigens exhibiting mitogenicity can cross-link cell membrane receptors on B and T lymphocytes leading to cell proliferation. All antigens vary greatly in physicochemical features such as biochemical nature, structural complexity, molecular size, foreignness, solubility, and so on. Objective: Thus, this review aims to describe the molecular bases of protein-antigenicity and those molecular bases that lead to an immune response, lymphocyte proliferation, or unresponsiveness. Conclusion: The epitopes of an antigen are located in surface areas; they are about 880-3,300 Da in size. They are protein, carbohydrate, or lipid in nature. Soluble antigens are smaller than 1 nm and are endocytosed less efficiently than particulate antigens. The more the structural complexity of an antigen increases, the more the antigenicity increases due to the number and variety of epitopes. The smallest immunogens are about 4,000-10,000 Da in size. The more phylogenetically distant immunogens are from the immunogen-recipient, the more immunogenicity increases. Antigens that are immunogens can trigger an innate or adaptive immune response. The innate response is induced by antigens that are pathogen-associated molecular patterns. Exogenous antigens, T Dependent or T Independent, induce humoral immunogenicity. TD protein-antigens require two epitopes, one sequential and one conformational to induce antibodies, whereas, TI non-protein-antigens require only one conformational epitope to induce low-affinity antibodies. Endogenous protein antigens require only one sequential epitope to induce cell-mediated immunogenicity.

Introduction: Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide with S-adenosine-L-methionine (SAM) as the methyl donor. Abnormal expression of NNMT is associated with many diseases (such as multiple cancers and metabolic and liver diseases), making NNMT a potential therapeutic target. Limited studies concerning the enzymesubstrate/ inhibitor interactions could be found to fully understand the detailed reaction mechanism. Methods: The binding affinity and ligand binding epitopes of nicotinamide or SAH for binding NNMT and its mutants were determined using saturated transfer difference (STD) nuclear magnetic resonance (NMR) techniques combined with site-directed mutagenesis. Results: The average dissociation constant of WT NNMT with nicotinamide and S-adenosine homocysteine (SAH) was 5.5 0.9 mM and 1.2 ± 0.3 mM, respectively, while the mutants Y20F and Y20G with nicotinamide were up to nearly 4 times and 20 times that of WT and with SAH nearly 2 times and 5 times that of WT. The data suggested that WT had the highest binding affinity for nicotinamide or SAH, followed by Y20F and Y20G, which was consistent with its catalytic activity. Conclusion: The binding affinity of nicotinamide and SAH to NNMT and its mutants were obtained by STD NMR in this study. It was found that nicotinamide and SAH bind to WT in a particular orientation, and Y20 is critical for their binding orientation and affinity to NNMT.

Background: Apelin-13 is an endogenous adipocytokine known for its antioxidant, antiinflammatory, and antiapoptotic properties. Objective: We aimed to investigate the possible protective effects of exogenous Apelin-13 administration on oxidative stress, inflammation, and apoptosis induced by the cytotoxic agent cyclophosphamide (CP) in the lungs. Methods: Twenty-four male Wistar albino rats were divided into four groups: Control (saline), CP (200 mg/kg), Apelin-13 (10 μg/kg/day), and CP+Apelin-13. CP was administered as a single dose on the fifth day, and apelin-13 was administered intraperitoneally for five days. Total oxidant status (TOS), total antioxidant status (TAS), and lipid peroxidation were determined with spectrophotometry, TNFα and IL1β were determined with ELISA, APJ, Sirt1, NF-ΚB, and p53 mRNA expressions were determined with qRT-PCR, cytochrome (Cyt) C and caspase-3 protein expressions were studied with western blotting in lung tissues. The oxidative stress index (OSI) was also calculated. Furthermore, serum surfactant protein-D (SP-D) and Krebs von den Lungen-6 (KL-6) levels were measured with ELISA. Results: Compared to the control group, TOS, OSI, lipid peroxidation, TNFα, IL1β, cyt C, caspase-3, APJ, NF-ΚB, and p53 were higher, and Sirt1 was lower in the lung tissue of rats in the CP group. Serum KL-6 and SP-D levels were higher in the CP group. Co-administration of CP with Apelin-13 completely reversed the changes induced by CP administration. Conclusion: Exogenous Apelin-13 treatment protected lung tissue against injury by inhibiting cyclophosphamide-induced oxidative stress, inflammation, and apoptosis. This protective effect of apelin-13 was accompanied by upregulation of the Sirt1 and downregulation of NF-ΚB/p53 in the lungs.

Background: An increasing prevalence of biofilm forming strains by vancomycinresistance Staphylococcus aureus (VRSA) is one of the most important causes of antimicrobial resistance. VRSA possesses various regulatory factors to form and sustain biofilm in biotic or abiotic conditions. Among them, ureolytic activity is an important factor in the stabilization of biofilms by neutralizing the acidic environment. Various urease accessory proteins are required to activate the urease enzyme inside the biofilm. Objective: To optimize the cloning, expression and purification of urease accessory protein E from VRSA for determination of the secondary structure, and functional characterization by using Berthelot128;™s method. Methods: BAB58453.1 gene (which encodes possible urease accessory protein E), having 38% similarity to Bacillus pasteurii UreE protein, was cloned, expressed, and purified by single-step affinity chromatography for performing secondary structural studies using circular dichroism spectroscopy, and functional analysis using Berthelot128;™s and crystal violet assay. Results: Structure elucidation using NMR and circular dichroism spectroscopy techniques revealed that UreE protein has a partially foldedα-helical structure. Using Berthelot's method, it was identified that the purified UreE protein has enhanced urease enzyme activity, in comparison to the control. From the results of Berthelot128;™s and crystal violet assays, it was deduced that the selected gene (UreE protein) plays a key role in enhancing urease enzyme activity and contributes to biofilm stability. Conclusion: Structural studies on VRSA urease accessory proteins could aid in the identification of new drug targets or the development of effective antibiofilm strategies (in combination with other drug targets) against infections caused by biofilm-producing strains.

Background: AOS enzymes can be biochemical indicators of abnormal xylogenesis in Scots pine, and this mechanism has similar features with the metabolic base of abnormal xylogenesis in Karelian birch. Objective: AOS enzymes128;™ activity in 150-300-year-old Pinus sylvestris L. wood with straight-- grained wood and right-twisted spiral-grained wood, expressed in varying degrees (5-20 angle), grew in three sample plots in lingonberry and blueberry pine forest stands of different ages (100-300 years) in the middle taiga subzone in the Republic of Karelia. Methods: Plant tissues were ground in liquid nitrogen in a uniform mass and homogenized at 4°C in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2 and 0.5 mM PMSF. After 20 min extraction, the homogenate was centrifuged at 10000 g for 20 min (MPW-351R, Poland). The sediment was washed in the buffer thrice. The pooled supernatant and sediment were dialyzed at 4°C for 18-20 h against a tenfold diluted homogenization buffer. The enzymes' activity was determined spectrophotometrically (Spectrophotometer SF-2000, OKB Spectr, Russia). Proteins in the extracts were quantified by the method of Bradford. Results: The study showed that the activity of SS, ApInv, CAT, POD and PPO in xylem and PPO in phloem were biochemical indicators for abnormal wood of P. sylvestris. We noticed an increase in sucrose metabolism in the apoplast and the activity of POD and PPO under spiral-grain wood formation like under figured wood formation earlier. We assume that the alternative pathway of sucrose metabolism (an indicator of abnormal xylogenesis in B. pendula var. carelica plants) that lead to restructuring of AOS enzymes have the same biochemical regularities in the spiral-grain wood formation in P. sylvestris. Conclusion: The study showed that the differences in the AOS enzyme's activity in P. sylvestris during the formation of straight-grained and spiral-grained wood were revealed for the first time. The increased CAT, POD and PPO activities in xylem with a decrease in SS and an increase in Ap- Inv during spiral-grained wood formation can be biochemical markers of these structural anomalies. Metabolic regularities found in the AOS enzyme complex during spiral-grained wood formation do not contradict those found earlier during figured wood formation in B. pendula var. carelica. The identified patterns can form the base for diagnostics of P. sylvestris wood quality in forest seed plantations and in their natural growth, which is necessary both for fundamental science and in various industry areas while high-quality material harvesting.

Background: Management of gluten intolerance is currently possible only by consumption of a gluten-free diet (GFD) for a lifetime. The scientific community has been searching for alternatives to GFD, like the inclusion of natural proteases with meals or pre-treatment of gluten-containing foods with glutenases. Actinidin from kiwifruit has shown considerable promise in digesting immunogenic gliadin peptides compared to other plant-derived cysteine proteases. Methods: In this study, we aimed to understand the structural basis for the elevated protease action of actinidin against gliadin peptides by using an in silico approach. Results: Docking experiments revealed key differences between the binding of gliadin peptide to actinidin and papain, which may be responsible for their differential digestive action. Conclusion: Sequence comparison of different plant cysteine proteases highlights amino acid residues surrounding the active site pocket of actinidin that are unique to this molecule and hence likely to contribute to its digestive properties.

Background: BSN-37, a novel antimicrobial peptide (AMP) containing 37 amino acid residues isolated from the bovine spleen, has not only antibacterial activity but also immunomodulatory activity. Recent evidence shows that long non-coding RNAs (lncRNAs) play an important role in regulating the activation and function of immune cells. The purpose of this experiment was to investigate the lncRNA and mRNA expression profile of mouse macrophages RAW264.7 stimulated by bovine antimicrobial peptide BSN-37. Methods: The whole gene expression microarray was used to detect the differentially expressed lncRNA and mRNA between antimicrobial peptide BSN-37 activated RAW264.7 cells and normal RAW264.7 cells. KEGG pathway analysis and GO function annotation analysis of differentially expressed lncRNAs and mRNA were carried out. Eight kinds of lncRNAs and nine kinds of mRNA with large differences were selected for qRT-PCR verification, respectively. Results: In the current study, we found that 1294 lncRNAs and 260 mRNAs were differentially expressed between antibacterial peptide BSN-37 treatment and control groups. Among them, Bcl2l12, Rab44, C1s, Cd101 and other genes were associated with immune responses and were all significantly up-regulated. Mest and Prkcz are related to cell growth, and other genes are related to glucose metabolism and lipid metabolism. In addition, some immune-related terms were also found in the GO and KEGG analyses. At the same time, real-time quantitative PCR was used to verify selected lncRNA and mRNA with differential expression. The results of qRT-PCR verification were consistent with the sequencing results, indicating that our data were reliable. Conclusion: This study provides the lncRNA and mRNA expression profiles of RAW264.7 macrophages stimulated by antimicrobial peptide BSN-37 and helps to provide a reference value for subsequent studies on lncRNA regulation of antimicrobial peptide BSN-37 immune function.