Protein and Peptide Letters - Volume 29, Issue 11, 2022

Background: Zika fever affects poor and vulnerable populations, presenting cycles observed in, at least 86 countries, with no vaccine prevention or treatment available. It is known that the genus Flavivirus causes Zika Virus (ZIKV), as Dengue and Yellow Fever, whose genetic material decodes, among other proteins, a series of non-structural (NS) proteins essential for viral replication, such as NS2B-NS3 protease. Additionally, chemical and biological systems are commonly studied using molecular modeling approaches allowing, among several other processes, to elucidate mechanisms of action, molecule reactivity and/or chemical properties and the design of new drugs. Thus, considering the in silico complexes between the biological target and the bioactive molecule, it is possible to understand better experimental results based on molecular properties, which are compared with the findings of the biological activity. Objective: Accordingly, this study aimed to present computational docking simulations of five previously reported active peptides against NS2B-NS3 protease of ZIKV and analyze some quantum chemical properties to identify the main contribution to improving the action. Methods: The compounds were described by Rut and coworkers (2017) and Hill and coworkers (2018), submitted to docking simulation in Gold software and quantum chemical properties calculations in Wavefunction Spartan software. Results: Total energy, electrophilicity index (ω) and energy gap (GAP) appeared to be the best properties to justify the peptide's biological activity. Moreover, the most promising compound (P1, Km 4.18 μM) had the best value of total energy (- 2763.04001 au), electrophilicity index (8.04 eV) and GAP (6.49 eV), indicating an energetically favorable molecule with good interaction with the target and, when compared to other peptides, presented moderate reactivity. P4 showed the highest electrophilicity index value (28.64 eV), which justified the interaction ability visualized in the docking simulation. However, its GAP value (4.24 eV) was the lowest in the series, suggesting high instability, possibly validating its low biological activity value (Km 19 uM). GAP was important to understand the chemical instability, and high values can promote damage to biological response. Conclusion: Furthermore, it was also noted that high electron affinity, related to the electrophilicity index, promoted electron-accepting characteristics, which was important to improve the biological activity of the peptides. A larger compound series must be studied to access features more precisely. However, these results have paramount importance in guiding future effort in this extremely-need health area.

Background: In recent years, cell pyroptosis has made it widely concerned. Pyroptosis is characterized by the activation of pathways leading to the activation of NLRP3 inflammasome and its downstream effector, such as interleukin (IL)-1β and IL-18, which has close relationship with inflammation. Recent evidence supports that CoenzymeQ10 (CoQ10) reduces related inflammatory factors (NLRP3, IL-1β and IL-18), which are associated with cell pyroptosis. This paper reviews the possible mechanisms of CoQ10 inhibiting pyroptosis of different cells and its possible mechanism. Further research is needed to better define the response effects of CoQ10 on specific aspects of cell pyroptosis (such as priming, promotion, and signaling), and to further investigate the organizational and cellular mechanisms by which CoQ10 reduces pyroptosis in different cells.

Background: MicroRNA-490-3p (miR-490-3p) plays a role in the pathogeneses of a variety of cardiovascular diseases. Bioinformatic analysis showed that miR-490-3p was downregulated in the myocardial tissues of mice with myocardial infarction (MI). Nevertheless, the functions and mechanisms of miR-490-3p in MI remain unclear. Methods: This study used an in-vitro model to investigate the role of miR-490-3p in MI. Human cardiac myocytes (HCMs) were cultured in a hypoxic environment. 3-(4,5)-Dimethylthiahiazo (-zy1)- 3,5-di-phenytetrazoliumromide (MTT) assay and flow cytometry were used to detect cell viability and apoptosis. The expression levels of forkhead box O1 (FOXO1) and miR-490-3p were detected by quantitative real-time PCR and Western blot. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), lactate dehydrogenase (LDH), cardiac troponin I (cTnI), and creatine kinase MB (CK-MB) were detected by enzyme-linked immunosorbent assay (ELISA). The targeted relationship between miR-490-3p and FOXO1 3’UTR was determined by a dual-luciferase reporter gene assay. Results: miR-490-3p was significantly down-regulated in hypoxia-induced HCM cells, while FOXO1 was markedly up-regulated. miR-490-3p overexpression inhibited HCM cell inflammatory responses and injury after hypoxia treatment. FOXO1 was validated to be a direct target of miR- 490-3p, and its overexpression weakened the effects of miR-490-3p on cell viability, apoptosis, as well as inflammatory responses. Conclusion: miR-490-3p alleviates cardiomyocyte injury via targeting FOXO1 in MI.

Background: An antigen is a small foreign substance, such as a microorganism structural protein, that may trigger an immune response once inside the body. Antigens are preferentially used rather than completely attenuated microorganisms to develop safe vaccines. Unfortunately, not all antigens are able to induce an immune response. Thus, new adjuvants to enhance the antigen’s ability to stimulate immunity must be developed. Objectives: Therefore, this work aimed to evaluate the molecular-structure adjuvant activity of tannic acid (TA) coupled to a protein antigen in Balb/c mice. Methods: Bovine serum albumin (BSA) was used as an antigen. The coupling of BSA and TA was mediated by carbodiimide crosslinking, and verified by SDS-PAGE. Forty-two Balb/c mice were divided into seven groups, including two controls without antigen, an antigen control, an adjuvant control, and two treatment groups. An additional group was used for macrophages isolation. A 30-day scheme was used to immunize the mice. The analysis of humoral immunity included immunoglobulin quantification, isotyping and antigen-antibody precipitation. The analysis of cell-mediated immunity included the quantification of nitric oxide from peritoneal macrophages and splenocytes’ proliferation assay after treatment stimulation. Results: No differences were found in the antibodies’ concentration or isotypes induced with the conjugate or the pure BSA. However, an immunogenicity improvement (p < 0.05) was observed through the specific anti-BSA antibody titers in mice immunized with the conjugate. Besides, macrophage activation (p < 0.05) was detected when stimulated with the treatments containing TA. Conclusion: Tannic acid exhibited macrophages’ activation properties. Moreover, when TA was incorporated into the structure of a protein antigen, such as BSA, an antibody specificity enhancement was observed. This was a consequence of antigen processing by activated antigen-presenting cells. These results showed the use of tannic acid as a novel candidate for vaccine molecular-structure adjuvant.

Background: Diabetes mellitus, a common metabolic disorder that causes high blood glucose, is due to impaired insulin secretion. Prolonged high blood sugar is associated with heart disease. Many proteins are involved in metabolic pathways and contractility of cardiac cells regulate cardiac hypertrophy, altering normal cardiac physiology and function. Moreover, microRNAs are essential regulators of these proteins. Thus, there is a need to study the protein and microRNA alterations in cardiomyocytes to better understand the mechanisms activated during cardiac stress. Objective: The study aims to profile differentially expressed sarcomere proteins in H9C2 cell lines under high glucose conditions compared with normal conditions, along with the identification of miRNAs regulating these proteins. Methods: Cardiac myoblast cell lines were treated with D-Glucose at three concentrations (10 mM, 25 mM, and 50 mM). Total cell protein was analyzed by Tandem Mass spectrometry Nano LCMS/ MS. Furthermore, next-generation sequencing data were analyzed for detecting microRNAs regulating cardiac cell protein expression. Bioinformatics databases such as Uniprot, Ingenuity Pathway Analysis (IPA), PANTHER, and Target scan were used. Results: The Nano LC-MS/MS analysis showed 2891 protein, 1351 protein groups, and 4381 peptide groups in both glucose-treated and control samples. Most proteins were metabolite interconversion enzymes, translation proteins, and proteins regulating the cytoskeleton. IPA analysis revealed differentially expressed proteins involved in EIF2 signaling, actin cytoskeleton signaling, cardiac fibrosis, and cell death. Moreover, the proteins troponin, tropomyosin, myosin, alpha-actin, and ATP synthase were found to be downregulated, thus responsible for altering sarcomere protein expression. Rno-mir-92b-5p was observed to be highly upregulated at 50 mM. Its target genes namely TPM2, ATP1A2, and CORO1C were mostly components of the sarcomere complex and its regulators. Conclusion: A combination of proteomic profile and microRNA profile of hyperglycemic cells provides an insight into advanced therapeutics. Our study has highlighted the role of sarcomere proteins, activation of Eukaryotic Initiation Factor 2 (EIF2) signaling, and suppression of actin cytoskeleton signaling in the pathophysiology of cardiomyopathy. MiR-92b-5p has an important role in regulating sarcomere protein complex activated.

Background: 3α-Hydroxysteroid dehydrogenase (3α-HSDH) reversibly catalyzes the oxidation of the C3-hydroxyl group of steroids, and has been used in clinical applications to detect serum total bile acid (TBA). In this study, A novel 3α-HSDH (called Sb 3α-HSDH) was expressed and characterized. Methods: Plasmid pGEX-6p-1 was used for the expression of Sb 3α-HSDH in Escherichia coli (BL21), and activities were determined by recording the change in absorbance at 340 nm with/without adding of ions. A prediction of its three-dimensional structure was performed with AlphaFold. Results: The substrate specificity test indicated that Sb 3α-HSDH is NAD(H)-dependent and has no activity with NADP(H). We also showed that Sb 3α-HSDH can catalyze the oxidation reaction of GCDCA and GUDCA with catalytic efficiencies (kcat/Km) of 29.060 and 45.839 s^-1mM^-1, respectively. The temperature dependence of catalysis suggests that Sb 3α-HSDH is a member of the mesophilic enzymes with its best activity at about 45 °C. The optimum pH of Sb 3α-HSDH was found to be between pH 8.0 and 9.0. The effect of ions, including K⁺, Mg²⁺, Na⁺, Cu²⁺, Mn²⁺, Fe²⁺, and Fe³⁺ on enzyme activity was evaluated and K⁺ and Mg²⁺ were found to enhance the activity of Sb 3α-HSDH by about 20% at concentrations of 200 mM and 50 mM, respectively. The well-conserved GIG motif, the active sites, and the Rossmann fold in the threedimensional structure indicate that Sb 3α-HSDH belongs to the “classical” type of SDR superfamily. Conclusion: We expressed and characterized a novel NAD(H)-dependent 3α-HSDH with typical threedimensional characteristics of the SDRs that exhibited substrate specificity to GCDCA and GUDCA.

Background: Premature ovarian insufficiency (POI) is a defect of ovarian functions in women younger than 40 years old. Although a large number of studies have focused on investigating autoimmune POI, its detailed pathogenesis is still largely unknown. Several studies have indicated that Myrcene exerted a part in the biological processes of various diseases. Nonetheless, whether Myrcene could influence the development of autoimmune POI remains to be elucidated. Methods: POI model was established by injecting zona pellucida glycoprotein 3 (pZP3). Hematoxylin and eosin (H) staining was applied to evaluate the pathological features of ovarian tissues. Enzymelinked immunosorbent assay (ELISA) was used for assessing the concentrations of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH) and interleukin (IL)-17. Flow cytometry analysis was conducted for assessing the balance of Th17/Treg cells. Results: The results showed that decreased levels of body weight, ovarian weight and ovarian index were reversed by Myrcene in POI model mice. The estrous cycles in mice were extended in pZP3 mice and Myrcene administration restored it to normal. The reduced number of primordial, primary, and secondary follicles as well as the increased number of atretic follicles in POI mice were offset by Myrcene administration. Moreover, Myrcene could modulate the Th17/Treg balance in autoimmune POI. Besides, Myrcene suppressed the MAPK signaling pathway in pZP3 mice. Conclusion: Myrcene regulated the Th17/Treg balance and endocrine function in autoimmune POI mice through the MAPK signaling pathway, which might provide a reference for improving the treatment of autoimmune POI.

Background: Ischemia-reperfusion (IR) injury is one of the major causes of acute kidney injury (AKI). Chemerin chemokine-like receptor 1 (CMKLR1) has been reported to be involved in the progression of IR injury. Here, we investigated the protective role of CMKLR1 antagonist, α-NETA, in IR mouse model, and dissected the underlying regulatory mechanism. Methods: IR injury mouse model was established to evaluate the protective effects of α-NETA on IR injury. Kidney injury-associated parameters and functions were examined to evaluate the renal function of Sham, IR, and IR+ α-NETA mice. Renal morphological changes and apoptosis were determined by PAS and TUNEL staining in IR and α-NETA treated mice. ELISA, RT-qPCR, and western blot were performed to examine the inflammatory responses and expression of CMKLR1. Results: α-NETA administration attenuated IR-induced renal tubular injury and epithelial cell apoptosis in IR injury mice. Kidney injury-related cystatin C, kidney injury molecule-1, neutrophil gelatinaseassociated lipocalin, and renal morphology were significantly improved. Mechanistically, α-NETA suppressed the inflammatory responses by inhibiting the expression of CMKLR1, and then protected the IR-induced renal damage and restored renal function. Conclusion: CMKLR1 plays an important role in renal ischemia-reperfusion injury, targeting CMKLR1 by using the small molecule inhibitor α-NETA is a potential treatment strategy for AKI.

Background: Hoatz is a vertebrate-specific gene, the defects of which result in hydrocephalus and oligo-astheno-teratozoospermia in mice. It encodes a 19-kDa protein lacking any domains of known function. Methods: To understand the protein activity, we purified the carboxyl-terminal fragment that is conserved among different species, and analyzed its structure and potential binding proteins. A soluble 9.9-kDa HOATZ fragment, including a poly-histidine tag (designated HOATZ-C), was purified to homogeneity. Results: The gel filtration profile and circular dichroism spectra collectively indicated that HOATZ-C was intrinsically disordered. When HOATZ-C was mixed with cleared lysate from Hoatz-null mouse testis, several proteins, including two of ~70 kDa size, were specifically co-purified with HOATZ-C on a nickel column. Conclusion: Based on the peptide mass fingerprinting of these bands, two members of the heat-shock protein family A were identified. These data may indicate the role of HOATZ in stress regulation in cells characterized by motile cilia and flagella.

Background: Worldwide, type 2 diabetes mellitus accounts for a considerable burden of disease, with an estimated global cost of >800 billion USD annually. For this reason, the search for more effective and efficient therapeutic anti-diabetic agents is continuing. Recent studies support the search for coumarins or related compounds with potential blood glucose-lowering properties. Aim: The study aims to design, synthesize and evaluate the hypoglycemic activity of a new class of 7-hydroxy coumarin derivatives. Objective: To explore and establish the in-silico-driven pharmacological role of a new class of 7- hydroxy coumarin derivatives as the therapeutic strategies against type 2 diabetes mellitus. Methods: A new class of 7-hydroxy coumarin derivatives was designed by assessment of their physicochemical properties and molecular docking against the Glucagon-like peptide-1 (GLP-1) receptor. Two novel series of 30 compounds were synthesized. The chemical structures of all the synthesized analogues have been elucidated by spectral studies of IR, ¹H-NMR, and mass spectroscopy. After considering the molecular docking score and their physicochemical properties, the compounds were screened out for the evaluation of their hypoglycemic potential. The compounds were investigated for their hypoglycemic activity using a streptozotocin (STZ) induced diabetic model and an oral glucose tolerance test (OGTT) method at different dose levels. Results: The molecular docking studies of synthesized derivatives reveal significant molecular interaction with the various amino acid residues of the GLP-1 receptor. IR spectral analysis revealed a strong band of -NH stretching in the range of 3406.7-3201.61 cm^-1 and one strong band for the lactone carbonyl group of the coumarin ring in the range of 1722.0-1703.5 cm^-1, confirming the chemical structure of all produced compounds. The synthesized coumarin analogues with the best docking score exhibited remarkable hypoglycemic potential as assessed by the STZ model and the OGTT method. Conclusion: Coumarin derivatives explored a good structure-activity relationship (SAR) and produced significant hypoglycemic potential.

Background: Alzheimer’s disease (AD) is characterized by the aggregation of Tau protein and Amyloid-β peptides (Aβ 1-40 and Aβ 1-42). A loss of ribosomal population is also observed in the neurons in affected regions of AD. Our studies demonstrated that in vitro aggregation of amyloid forming proteins, Aβ peptides and Tau protein variants (AFPs), in the vicinity of yeast 80S ribosome can induce co-aggregation of ribosomal components. Objective: In this study, the ability of minute quantities of AFP-ribosome co-aggregates to seed the aggregation of a large excess of untreated 80S ribosomes was explored. Methods: The AFPs were purified using ion-exchange chromatography. Seeded aggregation of ribosomes in the presence of minute quantities of ribosome-protein co-aggregates or ribosomal components was studied using agarose gel electrophoretic and SDS-PAGE analysis of the pellets and Sucrose Density Gradient centrifugation of the supernatant obtained after centrifugation of the aggregation reaction mixture. Results: Our studies, therefore, demonstrate that minute quantities of AFP-80S co-aggregate have significant seeding potential and could lead to aggregation of a large excess of fresh 80S ribosomes and this seeding ability is sustained over multiple cycles of ribosome aggregation. The aggregation propensity of ribosomal components alone could contribute towards the seeding of ribosome aggregation. Conclusion: The ability of minute quantities of AFP-80S co-aggregates to seed the aggregation of a large excess of fresh 80S ribosomes would result in the loss of global ribosomal population in Alzheimer’s disease afflicted neurons. Hence, subject to further validation by in vivo studies, our in vitro studies indicate a significant mode of toxicity of amyloid aggregates that might be important in Alzheimer’s disease pathology.