Protein and Peptide Letters - Volume 30, Issue 2, 2023

Introduction: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained. Methods: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation. Results: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies. Conclusion: In particular, the flexible structure was induced by the A30P mutation.

Background: Small Ca²⁺-binding protein parvalbumin possesses two strong Ca²⁺/Mg²⁺- binding sites located within two EF-hand domains. Most parvalbumins have no tryptophan residues, while cod protein contains a single tryptophan residue, which fluorescence (spectrum maximum position and fluorescence quantum yield) is highly sensitive to the Ca²⁺ association/dissociation. Objective: Intrinsic protein fluorescence of cod parvalbumin can be used for elucidating the mechanism of Ca²⁺ binding to this protein. Fluorescence of the single tryptophan residue of cod parvalbumin has been used to monitor Ca²⁺-induced changes in the protein, both in steady-state and kinetic mode. Methods: Steady-state fluorescence spectra of cod parvalbumin were measured using Cary Eclipse spectrofluorimeter. Stopped-flow accessories in combination with a novel high-speed spectrofluorimeter were used for measurements of kinetics of Ca²⁺ dissociation from cod parvalbumin after fast mixing of Ca²⁺-loaded protein with a chelator of divalent metal cations ethylenediaminetetraacetic acid (EDTA). Results: The fluorescent phase plots (fluorescence intensity at a fixed wavelength plotted against a fluorescence intensity at another fixed wavelength), constructed from steady state and kinetical data, shows a break at [Ca²⁺]/[parvalbumin] ratio close to 1. This means that the transition passes through an intermediate state, which is a protein with one bound calcium ion. These observations indicate that the binding of Ca²⁺ to cod parvalbumin is sequential. Conclusion: The results of the present spectral study showed that the binding of Ca²⁺ to cod parvalbumin is a sequential process. Calcium dissociation rate constants for the two binding sites of cod parvalbumin evaluated from the kinetic data are koff1 = 1.0 s^-1 and koff2 = 1.5 s^-1.

Background: A major cause of economic losses in the medical implant sector has been bacterial biofilms due to their ability to persist on various surfaces and their tolerance against endogenous defences, antibiotics, or other anti-microbial agents. The quest for potential sources causing inhibition or disruption of bacterial biofilms has been taken up to alleviate the loss. Plantderived extracts such as essential oils, bioactive compounds and other solvent extracts are regularly being used instead of antibiotics and other synthetic compounds as they are safer, economical, and in many instances, have an elaborate history of traditional medicinal usage. Objectives: As a plant that has been traditionally used over the centuries, the Moringa oleifera Lam., or more commonly the drumstick tree, is being tapped for myriad pharmaceutical applications. The protein-rich leaf of this tree has not only proved to be of great nutritional value but also divulged numerous potential therapeutic applications. Methods: While reports of proteinaceous components are rare, here we report the efficacy of the aqueous extract of the leaf of M. oleifera and a 62 kDa protein component in the disruption of staphylococcal biofilms, which are largely implicated in nosocomial infections. Results: The application of the M. oleifera leaf extract protein had a marked effect on the biofilm growth or formation by Staphylococcus aureus. Conclusion: While the crude extract itself showed considerable disruption of biofilm formation, the application of the purified protein obtained after a two-step process led to a significant increase in the anti-biofilm activity.

Background: Polypeptides that comprise less than 100 amino acids (50 amino acids in some cases) are referred to as small proteins (SPs), however, as of date, there is no strict definition. In contrast to the small polypeptides that arise due to proteolytic activity or abrupt protein synthesis, SPs are coded by small open reading frames (sORFs) and are conventionally synthesized by ribosomes. Purpose of the Review: Although proteins that contain more than 100 amino acids have been studied exquisitely, studies on small proteins have been largely ignored, basically due to the unsuccessful detection of these SPs by traditional methodologies/techniques. Serendipitous observation of several small proteins and elucidation of their vital functions in cellular processes opened the floodgate of a new area of research on the new family of proteins called "Small proteins". Having known the significance of such SPs, several advanced techniques are being developed to precisely identify and characterize them. Conclusion: Bacterial small proteins (BSPs) are being intensely investigated in recent days and that has brought the versatile role of BSPs into the limelight. In particular, identification of the fact that BSPs exhibit antimicrobial activity has further expanded its scope in the area of therapeutics. Since the microbiome plays an inevitable role in determining the outcome of personalized medicine, studies on the secretory small proteins of the microbiome are gaining momentum. This review discusses the importance of bacterial small proteins and peptides in terms of their therapeutic applications.

Background: Solvent tolerance is a desired feature of microorganisms for their application in biotechnology. Organic solvent-tolerant microorganisms are able to thrive in the presence of organic solvents. Several mechanisms have been proposed to elucidate their intrinsic tolerance to organic solvents. Objective: The present review aims to summarize the state of the art of the roles of membrane proteins in microbial organic solvent tolerance. Strategies and challenges for improving the protective function of membrane proteins in organic solvent stress are also proposed. Results: Membrane proteins related to transporter, signal transduction, and material and energy metabolism are involved in solvent tolerance. Optimization of the expression level of membrane proteins and engineering of membrane proteins are utilized to tackle the toxicity caused by organic solvents. Conclusions: Membrane proteins occupy a strikingly important position in microbial solvent tolerance. Further research on novel methods in membrane proteins, trade-offs among overexpression and toxicity of membrane proteins and solvent yield, and a direct relationship between signaling pathways and solvent tolerance will advance the utilization of organic solvent-tolerant microorganisms in biotechnology.

Background: The SARS-CoV-2 pandemic originated in Wuhan, China in December 2019 and spread rapidly worldwide. The virus gets entry into target cells via angiotensin-converting enzyme 2 (ACE2) receptors and its gene is highly polymorphic. Introduction: The variations in SARS-CoV-2 susceptibility and severity can be explained on a genetic level by studying the polymorphism in ACE2 receptor polymorphism. Objective: A prospective case-control study was designed to compare the ACE2 levels in SARS-CoV- 2 patients with the healthy controls in the local population, for which a total of 100 EDTA-containing blood samples were included (50 SARS-CoV-2 IgM positive case and 50 healthy controls). Methods: PCR-RFLP was performed to investigate the polymorphism of ACE2 in genomic DNA and the ACE2 plasma levels were determined through ELISA. Results: No significant difference in allelic and genotype frequencies (GG, GA, AA) were observed while the ACE2 plasma levels were found to be decreased in positive samples. Conclusion: No significant association of the ACE2 gene polymorphism (G8790A) was found with the SARS-CoV-2 susceptibility in the Pakistani population which intimates the search for other genetic factors within the local population.

Background: Cytoskeletal elements play key roles in cell morphology, cell division, cell mobility, and DNA partitioning in all domains of life. The IF-like protein FilP was discovered in Streptomyces coelicolor, and it was found to perform a structurally important cytoskeletal role by providing direct mechanical support for the cells. Objective: This work investigated the factors influencing FilP polymerization under a variety of conditions. Methods: DLS technique was applied to real-time monitor the in vitro assembly process of Streptomyces coelicolor FilP. Results: The presence of small amounts of divalent cations, such as CaCl2 or MgCl2, enhanced the polymerization of FilP, while higher amounts suppressed its polymerization. Moreover, high concentrations of NaCl, KCl, NH4Cl, and KNO3 both suppressed the polymerization of FilP. EDTA was found to have a very prohibitive effect on FilP polymerization, and even the following addition of Ca²⁺ could not initiate the assembly of FilP. FilP polymerized under a range of pHs ranging from pH 6 to pH 8, while the polymerization degree was sensitive to pH. FilP formed network-like, striated filaments at neutral pH, while the filaments became more disordered or loosely packed at pH 8 and pH 6, respectively. Conclusion: FilP assembly is calcium-mediated. Ca²⁺ is not only required for FilP polymerization, but also required for FilP to maintain the higher-order polymer structure. The accelerative effect of Ca²⁺ and the suppressive effect of Na⁺ persisted under a wide range of conditions, suggesting that FilP might use calcium and sodium ions as a general mechanism to mediate its polymerization process.

Background: Cervical carcinoma (CC) is the third most common cancer among females and the fourth leading cause of cancer-related death, which poses a serious threat to women's health. This study investigated the biological function and mechanism of circRNA circ_0002762 in the malignant progression of CC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify circ_0002762, microRNA-375 (miR-375) and Y-box binding protein 1 (YBX1) mRNA expressions in CC tissues and cell lines. After circ_0002762 was overexpressed in CC cell lines, cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) and wound healing assays were executed to probe cell growth and migration. Additionally, the targeting relationships between miR-375 and circ_0002762 or YBX1 3’-UTR were confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Western blot was adopted to examine YBX1 protein levels in CC cells. Results: Circ_0002762 expression was raised in CC tissues and cell lines, and highly expressed circ_0002762 was associated with larger tumor size and lymph node metastasis of CC patients. Circ_0007262 overexpression markedly accelerated the proliferation and migration of CC cells. Besides, miR-375 was revealed to be a downstream target of circ_0002762, and miR-375 overexpression counteracted the promoting effects of circ_0002762 overexpression on CC cell viability and migration. YBX1 was identified as a target of miR-375, and circ_0002762 positively modulated YBX1 expressions through adsorbing miR-375. Conclusion: Circ_0002762 promotes the progression of CC via sponging miR-375 and up-regulating YXB1 expression.

Background: Allergic diseases induced by dust have seriously threatened human health, while Bactrian camels can live in a sandy environment for a long time. Objective: To prepare rabbit anti-Bactrian camel IgE antibody and explore the distribution characteristics of IgE⁺ secretory cells in the palatine tonsils, which lays a theoretical foundation for the distribution of local antibodies in the palatal tonsils of Bactrian camel and the study of immune function. Methods: In this study, the amino acid sequences of Bactrian camel IgE, IgA, IgM and IgG heavy chain constant regions were compared, and a specific IgE gene fragment were selected (447 bp). The recombinant plasmid pET-28a-IgE was induced in Escherichia coli BL21(DE3) by IPTG and its expression conditions were optimized. The antibody was prepared by immunizing rabbits with purified IgE recombinant protein, its titer and specificity were detected by indirect ELISA and Western blotting. Immunohistochemical and statistical methods investigated the distribution of IgE⁺ secretory cells in the palatine tonsils. Results: The IgE recombinant protein was expressed in the form of inclusion bodies with a size of 16 kDa. The optimal IPTG induction concentration was 0.7 mmol/L and the induction time was 8 h. The titer of the antibody was 1:16000 by ELISA, and the antibody could specifically bind to the recombinant protein by Western blotting. IgE⁺ secretory cells were mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil of the Bactrian camel, followed by the subepithelial compartments of stratified squamous epithelium and occasionally in the extrafollicular region. Conclusion: The rabbit anti-Bactrian camel IgE polyclonal antibody was successfully prepared. It is confirmed that IgE exists in the palatine tonsils of Bactrian camels under normal living conditions. In addition, IgE⁺ secretory cells are mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil, which is consistent with the distribution characteristics of IgG⁺ and sIgA⁺ secretory cells in the palatal tonsils of the Bactrian camel.

Background: Temporin is one family of the shortest antimicrobial peptides found in Ranidae frogs. Staphylococcus aureus is one of the main pathogens of suppurative diseases and food contamination, causing severe local or systemic infections in humans. Temporin-GHa (GHa) was previously obtained from Hylarana guentheri, showing weak antibacterial activity against S. aureus. Most temporin peptides are positively charged by arginine and lysine; however, GHa contains histidine. Objective: In order to investigate the impact of positively charged amino acid on its antibacterial and antibiofilm activity, GHa4R was designed and synthesized by replacing histidine with arginine in GHa. Methods: The antibacterial activity and efficacy against S. aureus were detected by minimum inhibitory concentration, minimum bactericidal concentration, and time-killing kinetics assays. The action mechanism was determined by propidium iodide uptake and scanning electron microscopy assays. The antibiofilm activity was measured by the MTT method. Eradication of biofilm was observed by fluorescence microscope. Results: Compared to GHa, GHa4R had stronger antibacterial activity and bactericidal efficacy against S. aureus. Impressively, GHa4R presented antibacterial activity against methicillin-resistant S. aureus (MRSA). It was barely affected by temperature, pH, and storage period, showing high stability. Furthermore, it increased the permeability of the cell membrane and damaged the membrane integrity, leading to cell death. In addition, GHa4R did not induce antibiotic resistance in S. aureus in 30 days, but the MIC of vancomycin was doubled. It not only inhibited S. aureus biofilm formation but also eradicated 24 h-biofilms. Conclusion: The above-mentioned characteristics make GHa4R a promising candidate for the treatment of S. aureus infections.