Protein and Peptide Letters - Volume 29, Issue 9, 2022

Bacillus subtilis is a Gram-positive bacterium that has gained an unprecedented reputation as an expression system at the industrial scale due to characteristics, such as GRAS (Generally recognized as safe), ease of genetic manipulation, high growth rate on the cheap substrate, and short fermentation cycle. This expression system has been widely accepted for the production of various chemicals, pharmaceutical products, food products, proteins, and enzymes. However, there are various hurdles to optimizing the production of heterologous protein in this expression system due to a lack of understanding regarding metabolic pathways and expression elements. In this review, we have emphasized strategies that can enhance the expression level of heterologous proteins in B. subtilis. These strategies include optimization of B. Subtilis strain, expression elements, such as promotors, UTR (Untranslated region), RBS (Ribosome binding site), signal peptide, and metabolic pathways. Finally, contemporary challenges and future perspectives of B. subtilis as an industrial-scale expression system are discussed.

Background: Tyrosinase inhibitor developments have been widely attended by investigators for their various applications. Objective: A combination of virtual screening of docking simulations and biochemical inhibition kinetics was performed to find a new inhibitor of tyrosinase for the clinical application of an antipigment agent. Methods: We conducted docking simulations to detect tyrosinase key binding residues and used the detected binding residues to screen the NCBI PubChem database for probing tyrosinase binding compounds. The serial inhibition kinetics and spectrofluorimetry measurements were performed to validate the inhibitory effect on tyrosinase. Results: We have detected 200 candidates and categorized them into four clusters. Among them, we successfully confirmed salsalate as a new inhibitor of tyrosinase measured by serial enzyme kinetics. Salsalate was detected as a reversible inhibitor of tyrosinase displaying a typical mixedtype inhibition manner (IC50 = 22.19 ± 1.01 mM; Ki = 19.98 ± 2.11 mM). Spectrofluorimetry measurement by integrating with 1-anilinonaphthalene-8-sulfonate showed that salsalate mainly induced a slight regional conformation distortion of the tyrosinase active site accompanied by a slight hydrophobic disruption. Conclusion: Our study suggests that salsalate is a potential anti-pigment drug via inhibition of tyrosinase activity and it might be applicable for dermatologic clinical application. Also, our study enlarges an insight into the salsalate drug application.

Background: This study aimed to investigate the effects of irisin on rat tracheal smooth muscle contraction-relaxation responses and the roles of voltage-gated potassium (KV) channels, ATP-sensitive potassium (KATP) channels, and large-conductance calcium-activated potassium (BKCa) channels in these effects. Methods: Isometric contraction and relaxation responses of tracheal segments were measured using the tissue bath method. Submaximal contractions were induced by ACh (10^-5 M) or KCl (60 mM), and then concentration-response curves of irisin (10^-9 to 10^-6 M) were obtained. For the temporal control, a double-distilled water group was formed. ACh and irisin were added to the baths after tracheal segments were incubated with 4-AP (KV channel blocker), glibenclamide (KATP channel blocker), TEA, and iberiotoxin (BKCa channel blockers) to assess the role of K+ channels. In addition, a vehicle group was performed for the solvent dimethyl sulfoxide (DMSO). Results: Irisin exhibited the relaxant effects in tracheal segments precontracted with both ACh and KCl at concentrations of 10^-8-10^-6 M (p<0.05). Besides, incubations of 4-AP, glibenclamide, TEA, and iberiotoxin significantly inhibited the irisin-mediated relaxation (p<0.05), whereas DMSO incubation did not modulate irisin responses (p>0.05). Conclusion: In conclusion, the first physiological results on the relaxant effects of irisin in rat trachea were obtained. Our findings demonstrated that irisin mediates concentration-dependent relaxation in rat tracheas. Moreover, the present study reported for the first time that irisin-induced bronchorelaxation is associated with the activity of the K+ channels.

Introduction: Protein S-nitrosylation (SNO) and O-GlcNAcylation are important posttranslational modifications. The biological connection between SNO and O-GlcNAcylation is not clear. Objective: We aim to identify the crosstalk between SNO and O-GlcNAcylation during heat-shock. Methods: Ex vivo heat-shock on mouse tissues together with in vitro heat-shock on culture cells was performed and global levels of SNO and O-GlcNAcylation were analyzed with Biotin-switch assay (BSA) and RL2 immunoblots. Results: Heat-shock induces hypo-SNO in parallel with hyper-O-GlcNAcylation. Inverted induction of hypo-SNO and hyper-O-GlcNAcylation is globally progressed in a time-dependent manner. Discussion: Moreover, heat-shock ubiquitously facilitates S-denitrosylation (SdeNO) of endogenous SNO-proteins including SNO-OGT, SNO-Hsp70, SNO-Hsp90, SNO-Akt, and SNOactin. Particularly, SdeNO of SNO-OGT leads to enhanced OGT activity. Conclusion: These findings provide mechanistic evidence that heat-shock triggers SdeNO of SNOOGT by which OGT activity is up-regulated, resulting in hyper-O-GlcNAcylation.

Background: Lectins are proteins with therapeutic and diagnostic potential that can be applied in battling various ailments. Aim and Objective: This study was designed to purify and characterize the hemagglutinating activity derived from the leaves of Calotropis procera and its possible role in protecting the stomach against ethanol-induced lesions. Methods: The Calotropis procera leaf lectin (ProLec), was isolated by homogenization of the defatted leaf powder in Phosphate-Buffered Saline (PBS) and purified by affinity chromatography on Sephadex G-100. The lectin was eluted from the affinity column by 3% acetic acid and was physicochemically characterized. In a dose-dependent manner, ProLec was administered to rats with ethanol-induced ulcers, and biochemical, histopathological, and toxicological examinations were performed. Results: ProLec is a heterodimer of 75 and 68 kDa. It agglutinated all human RBCs, whereas it showed weak interaction with animal erythrocytes. The protein was optimally active at 25 °C and was labile above this temperature. ProLec exhibited two pH optima and was a metalloprotein requiring Ca, Mn, and Ni. It contains 1.6% tryptophan residues of which about 1% is exposed and critical for lectin activity. The lectin exhibited a potent gastroprotective effect against ethanolinduced gastric lesions with no apparent toxicity to both kidneys and liver. Examination of the pH of the gastric juice of lectin-treated animals indicated a possible role of lectin in maintaining stomach acidity within the normal ranges compared to the gastric juice pH of animals that received ethanol only. Conclusion: These results may suggest that ProLec could conceivably be a good future drug for the treatment of gastric ulcers, however, extensive immunological and toxicological research remains to be done.

Background: Oral squamous cell carcinoma (OSCC) is one of the commonest malignancies of the oral cavity. FOXN3 is a tumor suppressor that represses the progression of many tumors. Nonetheless, its role in OSCC has not been elucidated. This work is performed to probe the role and dysregulation mechanism of FOXN3 in OSCC. Methods: FOXN3 mRNA and miR-299-5p expressions were quantified by quantitative real-time polymerase chain reaction (qRT-PCR); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to detect OSCC cell growth; Transwell experiment was conducted to detect cell migration and invasion; dual-luciferase reporter experiment and bioinformatics were adopted to analyze the relationship between miR-299-5p and FOXN3; Western blot was implemented to detect FOXN3 protein expression. Results: FOXN3 expression was remarkably down-modulated, and miR-299-5p expression was markedly up-modulated in OSCC tissues and cell lines compared with paracancerous tissues and normal oral epithelial cell line. FOXN3 overexpression impeded OSCC cell growth, migration and invasion. FOXN3 was proven to be a downstream target of miR-299-5p, and miR-299-5p mimics enhanced OSCC cell growth, migration and invasion. Moreover, FOXN3 overexpression partially reversed the promoting effects of miR-299-5p mimics on OSCC cell growth, migration and invasion. Conclusion: FOXN3 expression is remarkably down-modulated in OSCC tissues and cell lines, and miR-299-5p targets FOXN3 to facilitate OSCC cell growth, migration and invasion. These results imply that miR-299-5p/FOXN3 axis may be a potential target for OSCC treatment.

Objective: To probe the role of circular RNA_0008768 (circ_0008768) in the development of pancreatic cancer (PC) and its regulatory mechanism. Methods: The expression levels of circ_0008768, miR-330-3p, and PTEN mRNA in PC tissues and cells were detected using qRT-PCR. Cell proliferation, migration and invasion of PC cells were detected by CCK-8 method, EdU method, and Transwell assay. The targeting relationship between circ_0008768 and miR-330-3p, as well as miR-330-3p and PTEN mRNA 3'UTR was analyzed by the dual-luciferase reporter assay. PTEN expression levels in PC cells were analyzed by Western blot. Results: The expression levels of circ_0008768 and PTEN mRNA were significantly reduced in both PC tissues and cell lines. Overexpression of circ_0008768 exerted an inhibitory effect on the proliferation, migration and invasion of PC cells. Knocking down circ_0008768 showed the opposite effect. Circ_0008768 directly targeted and negatively regulated the expression of miR-330- 3p. PTEN was identified as a downstream target gene of miR-330-3p. Circ_0008768 could positively regulate the expression of PTEN. Conclusion: In PC, circ_0008768 can act as a tumor-suppressive factor to inhibit the development of PC by regulating the miR-330-3p/PTEN molecular axis.

Background: Annexin V, a member of calcium-dependent phospholipid-binding proteins, selectively binds to the exposed phosphatidylserine, which can be used for in vitro apoptosis detection. Simultaneous staining of cells with annexin V-fluorescein isothiocyanate (FITC) and the non-vital dye propidium iodide (PI) enables the detection of apoptotic and necrotic cells. Objective: Our study aimed to express, purify, and stabilize the recombinant annexin V. Methods: The recombinant annexin V was cloned and expressed in E. coli bacteria and was purified using Ni-IDA resin. The FITC conjugation was performed, and apoptosis detection of HaCaT cells by FITC-labeled annexin V was evaluated by flow cytometry. Then, the stability of FITC-labeled annexin in various conditions, including polyvinyl alcohol (PVA), glycerol, and trehalose, was evaluated. Results: The results showed that annexin V was appropriately expressed and purified. After FITC conjugation, it could perfectly detect the cell death of HaCat cells in different apoptosis percentages. FITC-labeled annexin had more stability with PVA than glycerol and trehalose. Conclusion: It seems that PVA has an acceptable effect on FITC-labeled annexin V stability in concentrations lower than 1 mg mL^-1 without interfering with fluorescent intensity.