Protein and Peptide Letters - Volume 31, Issue 2, 2024

Phage therapy, a promising alternative to combat multidrug-resistant bacterial infections, harnesses the lytic cycle of bacteriophages to target and eliminate bacteria. Key players in this process are the phage lysis proteins, including holin, endolysin, and spanin, which work synergistically to disrupt the bacterial cell wall and induce lysis. Understanding the structure and function of these proteins is crucial for the development of effective therapies. Recombinant versions of these proteins have been engineered to enhance their stability and efficacy. Recent progress in the field has led to the approval of bacteriophage-based therapeutics as drugs, paving the way for their clinical use. These proteins can be combined in phage cocktails or combined with antibiotics to enhance their activity against bacterial biofilms, a common cause of treatment failure. Animal studies and clinical trials are being conducted to evaluate the safety and efficacy of phage therapy in humans. Overall, phage therapy holds great potential as a valuable tool in the fight against multidrug- resistant bacteria, offering hope for the future of infectious disease treatment.

Introduction: Bacterial biofilm is known as the main cause of periodontal disease. Generally, the anaerobic Gram-negative, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are considered the most identified bacteria. Objective: This study aimed to investigate the antimicrobial effect and cytotoxicity of two experimental composites containing chitosan-silver oxide (CH-Ag2O) particles. Materials and methods: Four experimental groups, including Ag2O and CH, along with two composites of CH-Ag2O 20 and CH-Ag2O 60 mg, were prepared. Antimicrobial activity was performed against Porphyromonas gingivalis (ATCC#33277) and Fusobacterium nucleatum (ATCC#25586) using the agar dilution method. Moreover, the cytotoxicity assay was performed on human gingival fibroblasts (HGF) by the use of the MTT method. The obtained data were analyzed with descriptive methods, one-way ANOVA, and Tukey’s LSD tests. Results: The antibacterial activity of both composites was higher than both CH and Ag2O, and the greatest antibacterial properties were presented in CH-Ag2O 60. In all three measurements (24, 48, and 72 h), the greatest cytotoxicity was seen in Ag2O, followed by CH, CH-Ag2O 20, and CHAg2O 60 in descending order, respectively. The cytotoxicity of these components was related to the concentration and not to the time of exposure. The results showed that Ag2O in 3.7 and 7.5 μg/ml concentrations and CH-containing groups in 250 and 500 μg/ml were toxic to the cultured HGF. Conclusion: The experimental composite containing CH-Ag2O 60 showed the greatest antibacterial properties against two periodontal pathogens evaluated. In order to clarify the clinical significance of composite cytotoxicity, further clinical studies are necessary.

Background: Colorectal cancer (CRC) is a prevalent form of cancer globally, characterized by a high mortality rate. Therefore, discovering effective therapeutic approaches for CRC treatment is critical. Methods: The levels of KIF20A in CRC clinical samples were determined using Western Blot and immunofluorescence assay. SW480 cells were transfected with siRNA targeting KIF20A, while HT-29 cells were transfected with a KIF20A overexpression vector. Cell viability and apoptosis of CRC cells were assessed using CCK-8 and TUNEL analysis. Migration ability was investigated using Transwell. The levels of pyruvate, lactate and ATP were determined through corresponding assay kits. Western Blot was applied to confirm the level of proteins associated with glycolysis, c- Myc, HIF-1α, PKM2 and LDHA. Subsequently, functional rescue experiments were conducted to investigate further the regulatory relationship between KIF20A, c-Myc, and HIF-1α in colorectal cancer (CRC), employing the c-Myc inhibitor 10058-F4 and c-Myc overexpression plasmids. Results: KIF20A was up-regulated in vivo and in vitro in CRC. KIF20A knockdown inhibited cell viability and migration while promoting cell apoptosis in SW480 cells. Conversely, overexpression of KIF20A yielded contrasting effects in HT-29 cells. Moreover, inhibition of KIF20A restrained the pyruvate, lactate production and ATP level, whereas overexpression of KIF20A enhanced the Warburg effect. Western Blot indicated that knockdown KIF20A attenuated the levels of c-Myc, HIF-1α, PKM2 and LDHA. In addition, rescue experiments further verified that KIF20A enhanced the Warburg effect by the KIF20A/c-Myc/HIF-1α axis in CRC. Conclusion: KIF20A, being a crucial regulator in the progression of CRC, has the potential to be a promising therapeutic target for the treatment of CRC.

Background: Dexmedetomidine (Dex) is widely used in perioperative anesthesia, and recent studies have reported that it protects organs from ischemia/reperfusion (I/R) injury. Objectives: This study was performed to investigate the role of Dex in alleviating cerebral I/R injury and its regulatory effects on metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-140-5p (miR-140-5p)/nuclear factor erythroid-derived 2-like 2 (Nrf2) axis. Methods: In vivo rat middle cerebral artery occlusion (MCAO) model and in vitro oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced neuronal injury model were constructed. Dex was injected into the animals or used to culture HT22 cells to observe the pharmacological effects. The neurological defect, brain water content, infarct volume of the rats, and neuron viability were evaluated. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were detected. Besides, the regulatory effects of Dex on MALAT1, miR-140-5p, and Nrf2 expression levels and regulatory relationships among them were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and dual- luciferase reporter assay. Results: Dex significantly alleviated the neurological injury of rats with MCAO and promoted the viability of neurons. Dex treatment suppressed miR-140-5p expression, but elevated MALAT1 and Nrf2 expressions. MALAT1 knockdown down-regulated Nrf2 expression and promoted oxidative stress in neurons. Additionally, miR-140-5p directly targeted Nrf2, and it also functioned as a downstream target miRNA of MALAT1. Conclusion: Dex, via regulating MALAT1/miR-140-5p/Nrf2 axis, plays a neuroprotective role against I/R-induced brain injury.

Background: Binding appropriate cellular receptors is a crucial step of a lifecycle for any virus. Structure of receptor-binding domain for a viral surface protein has to be determined before the start of future drug design projects. Objectives: Investigation of pH-induced changes in the secondary structure for a capsid peptide with loss of function mutation can shed some light on the mechanism of entrance. Methods: Spectroscopic methods were accompanied by electrophoresis, ultrafiltration, and computational biochemistry. Results: In this study, we showed that a peptide from the receptor-binding domain of Parvovirus B19 VP1 capsid (residues 13-31) is beta-structural at pH=7.4 in 0.01 M phosphate buffer, but alpha- helical at pH=5.0, according to the circular dichroism (CD) spectroscopy results. Results of infra- red (IR) spectroscopy showed that the same peptide exists in both alpha-helical and beta-structural conformations in partial dehydration conditions both at pH=7.4 and pH=5.0. In contrast, the peptide with Y20W mutation, which is known to block the internalization of the virus, forms mostly alpha-helical conformation in partial dehydration conditions at pH=7.4. According to our hypothesis, an intermolecular antiparallel beta structure formed by the wild-type peptide in its tetramers at pH=7.4 is the prototype of the similar intermolecular antiparallel beta structure formed by the corresponding part of Parvovirus B19 receptor-binding domain with its cellular receptor (AXL). Conclusion: Loss of function Y20W substitution in VP1 capsid protein prevents the shift into the beta-structural state by the way of alpha helix stabilization and the decrease of its ability to turn into the disordered state.

Background: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue. Objectives: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in Escherichia coli expression system. Results: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of β-Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system. Conclusion: Antimicrobial peptides Histatin 1, β -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the Escherichia coli expression system.

Background: 7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays a pivotal role in vivo in the biotransformation of secondary bile acids and has great potential in industrial biosynthesis due to its broad substrate specificity. In this study, we expressed and characterized a novel thermostable 7α-HSDH (named Sa 7α-HSDH). Methods: The DNA sequence was derived from the black bear gut microbiome metagenomic sequencing data, and the coding sequence of Sa 7α-HSDH was chemically synthesized. The heterologous expression of the enzyme was carried out using the pGEX-6p-1 vector. Subsequently, the activity of the purified enzyme was studied by measuring the absorbance change at 340 nm. Finally, the three-dimensional structure was predicted with AlphaFold2. Results: Coenzyme screening results confirmed it to be NAD(H) dependent. Substrate specificity test revealed that Sa 7α-HSDH could catalyze taurochenodeoxycholic acid (TCDCA) with catalytic efficiency (kcat/Km) 3.81 S-1 mM-1. The optimum temperature of Sa 7α-HSDH was measured to be 75°C, confirming that it belongs to thermophilic enzymes. Additionally, its thermostability was assessed using an accelerated stability test over 32 hours. The catalytic activity of Sa 7α-HSDH remained largely unchanged for the first 24 hours and retained over 90% of its functionality after 32 hours at 50°C. Sa 7α-HSDH exhibited maximal activity at pH 10. The effect of metal ions-K⁺, Na⁺, Mg²⁺ and Cu²⁺-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg²⁺ was observed to enhance the enzyme’s activity by 27% at a concentration of 300 mM. Neither K⁺ nor Na+ had a significant influence on activity. Only Cu²⁺ was found to reduce enzyme activity. Conclusion: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.

Introduction: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. Objectives: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. Methods: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. Results: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. Conclusion: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.