Protein and Peptide Letters - Volume 29, Issue 10, 2022

Ciliary neurotrophic factor (CNTF) is a pluripotent neurotrophic factor originally isolated from chicken embryo ciliary neurons. It has a powerful role in developing and maintaining the optic nervous system and has been used for many vision-related diseases. It also plays an important role in the neurogenesis, regeneration and survival of other neurons, including neural stem cells, dorsal root ganglion, sensory neurons and motor neurons. CNTF is related to neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. In addition to its role in the nervous system, CNTF regulates the balance of energy metabolism and the administration of CNTF induces body weight loss. More CNTF functions have been found with the deepening of study, such as protecting and promoting cardiomyocyte proliferation. In addition, CNTF even participates in mental illness and inflammation suppressing. CNTF exerts multidirectional physiological activity by regulating the transcription of various genes through a variety of signalling pathways (including JAK/STAT, MAPK, and PI3K/AKT). This review summarizes the roles and mechanisms of CNTF in the optic nervous system, retinal-related diseases, neuronal protection, and especially nutrition, energy metabolism and other aspects.

Background: Ovarian carcinoma (OC) is one of the most common malignancies of the female reproductive organs, with a low survival rate primarily due to the lack of effective methods for early diagnosis and prognosis. Objective: In this article, our motivation is to explore the lncRNA-related network mechanisms involved in the pathogenesis of OC. Methods: Public lncRNAs and mRNA expression datasets for OC were collected from the Gene Expression Omnibus (GEO) database. By integrated bioinformatics analysis, we constructed a UCA1-miRNA-mRNA network. We studied lncRNA-related molecular modulation mechanism in ovarian cancer cells based on MTT assay, dual luciferase reporter gene assays, quantitative realtime PCR, and western blotting. Results: UCA1 was higher in ovarian tumor tissues and cells than normal tissues and cells. It was demonstrated in this study that knockdown of UCA1 inhibited ovarian cancer cell viability, which a miR-99b-3p inhibitor could reverse in vitro. Further, UCA1 was shown to regulate the expression of SRPK1 by directly binding to miR-99b-3p. Conclusion: These results suggest that UCA1 functions as an oncogene in ovarian cancer. Inhibition of UCA1/miR-99b-3p/SRPK1 axis may become a novel target for treating ovarian cancer.

Background: The ESKAPE group of pathogens which comprise of multidrug resistant bacteria, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are the cause of deadly nosocomial infections all over the world. While these pathogens have developed robust strategies to resist most antibiotics, their ability to form biofilms is one of their most combative properties. Hence there is an urgent need to discover new antibacterial agents which could prevent or destroy the biofilms made by these bacteria. Though it has been established that lactoferrin (LF), a potent iron binding antibacterial, antifungal, and antiviral protein displays anti-biofilm properties, its mechanisms of action, in addition to its iron chelation property, still remains unclear. Objective: The binding and inhibition studies of LF with the enzyme Nucleoside diphosphate Kinase (NDK) and its elastase cleaved truncated 12 kDa fragment (12-NDK). Methods: The characterization studies of NDK and 12-NDK using florescence spectroscopy, dynamic light scattering, size exclusion chromatography and ADP-glo Kinase Assay. Inhibition studies of LF-NDK using ADP-glo kinase assay, Surface Plasmon Resonance and Biofilm inhibition studies. Results: NDK and 12-NDK were cloned, expressed and purified from Acinetobacter baumannii and Pseudomonas aeruginosa. The characterization studies revealed NDK and 12-NDK from both species are stable and functional. The inhibition studies of LF-NDK revealed stable binding and inhibition of kinase activity by LF. Conclusion: The binding and inhibition studies have shown that while LF binds with both the NDK and their truncated forms, it tends to have a higher binding affinity with the truncated 12 kDa fragments, resulting in their decreased kinase activity. This study essentially gives a new direction to the field of inhibition of biofilm formation, as it proves that LF has a novel mechanism of action in other than iron sequestration.

Objective: This study aimed to evaluate the combination of SDF-1 and KLD-12 to form self-assembling polypeptide and its effect on osteogenic differentiation. Methods: ELISA assay was performed to detect whether KLD-12 composite SDF-1 self-assembled polypeptide was successfully prepared. BMSCs were isolated and characterized by Flow cytometry. MTT assays, Calcein-AM/PI fluorescence staining, and Glycosaminoglycans (GAGs) measurement were carried out to detect cell viability after cells exposed to KLD-12 composite SDF-1 selfassembled polypeptide. The migration of cells induced by KLD-12 composite SDF-1 selfassembled polypeptide was also examined by transwell assay and Immunoblot. Osteogenic differentiation of cells stimulated with KLD-12 composite SDF-1 self-assembled polypeptide was analyzed by Immunoblot, Alizarin Red Staining, and Alkaline Phosphatase activity. Additionally, immunoblot and immunofluorescence assays were performed to investigate the effects of the polypeptide on the Wnt/β-catenin pathway. Results: KLD-12 composite SDF-1 self-assembled polypeptide was successfully prepared and identified. In addition, we isolated and characterized mouse mesenchymal stem BMSCs. Our data further revealed that KLD-12 combined with SDF-1 self-assembled polypeptide improved the survival of BMSCs and promoted cell migration. Moreover, the self-assembled polypeptide induced osteogenic differentiation of BMSCs. Mechanically, we found that the self-assembled polypeptide activated the Wnt/β-catenin pathway, therefore promoting the differentiation and migration of BMSCs. Conclusion: Our proposed treatment can potentially be effective for bone defects.

Background: Circular RNAs (circRNAs) exert an important regulatory effect on cancer progression. Reportedly, circRNAs can modulate gene expression by working as molecular sponges for miRNAs. Nonetheless, many functional circRNAs in hepatocellular carcinoma (HCC) remain to be identified. This study aimed to explore the role of hsa_circ_0006789 (circ_0006789) in HCC. Methods: The expression profile of circRNAs in HCC tumor tissues was analyzed using circRNA microarray data. Circ_0006789 expression in HCC tissues and cell lines was examined by qPCR. After circ_0006789 was overexpressed or knocked down in HCC cell lines, HCC cell growth, migration and invasion were evaluated by the CCK-8 method and Transwell experiment. RIP assay, RNA pull-down assay, dual-luciferase reporter experiment and Western blotting were adopted to investigate the regulatory mechanism among circ_0006789, microRNA (miR)-1324 and SRY (sex determining region Y)-box 12 (SOX12). Results: Circ_0006789 was overexpressed in HCC tissues and cell lines. Circ_0006789 overexpression accelerated the growth, migration and invasion of HCC cells, while knockdown of circ_0006789 exerted the opposite effects. miR-1324 was confirmed as a target of circ_0006789, and miR-1324 targeted SOX12 to suppress its expression. Circ_0006789 could promote SOX12 expression by sponging miR-1324. Conclusion: Circ_0006789 modulates the growth, migration and invasion of HCC cells by regulating miR-1324/SOX12 axis.

Background: Chitinases are plant defense-related proteins with a high biotechnological potential to be applied in agriculture. Objectives: This study aimed to purify a chitinase from the latex of Ficus benjamina. Methods: An antifungal class I chitinase, named FbLx-Chi-1, was purified from the latex of Ficus benjamina after precipitation with 30-60% ammonium sulfate and affinity chromatography on a chitin column and antifungal potential assay against phytopathogenic fungi important to agriculture. Results: FbLx-Chi-1 has 30 kDa molecular mass, as estimated by SDS-PAGE and the optimal pH and temperature for full chitinolytic activity were 5.5 and 60ºC, respectively. FbLx-Chi-1 is a high pH-, ion-tolerant and thermostable protein. Importantly, FbLx-Chi-1 hindered the growth of the phytopathogenic fungi Colletotrichum gloeosporioides, Fusarium pallidoroseum, and Fusarium oxysporum. The action mode of FbLx-Chi-1 to hamper F. pallidoroseum growth seems to be correlated with alterations in the morphology of the hyphal cell wall, increased plasma membrane permeability, and overproduction of reactive oxygen species. Conclusion: These findings highlight the biotechnological potential of FbLx-Chi-1 to control important phytopathogenic fungi in agriculture. In addition, FbLx-Chi-1 could be further explored to be used in industrial processes such as the large-scale environmentally friendly enzymatic hydrolysis of chitin to produce its monomer N-acetyl-β-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.

Background: A growing body of evidence demonstrates that miR-137 acts against cancers; however, the biological function of miR-137 in esophageal squamous cell carcinoma (ESCC) remains to be fully understood. Objective: The aim of this study is to explore the role of miR-137 in ESCC. Methods: miR-137 expression was detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and target protein expression was detected by western blot. Cell counting, colony formation and flow cytometry were employed to determine the effects of miR-137 on the growth of ESCC cells. Dual-luciferase reporter assay was performed to validate the binding of miR- 137 with a dishevelled-associated activator of morphogenesis 1 (DAAM1) 3’-UTR. Results: miR-137 was shown to be down-regulated in ESCC. miR-137 expression was inversely correlated with the 5-year survival rate of ESCC patients. Up-regulated miR-137 attenuated ESCC proliferation and promoted ESCC cell apoptosis. Meanwhile, to further reveal how miR-137 regulated the malignant behaviors of ESCC, the downstream mRNA binding targets of miR-137 were explored. miR-137 was demonstrated to bind DAAM1 3’-UTR and repressed the expression of DAAM1. The expression of DAAM1 and miR-137 in ESCC was inversely correlated. Additionally, the reintroduction of DAAM1 had the capacity to reverse the negative role of miR- 137 in ESCC cell growth. Conclusion: These findings have uncovered the new function of miR-137 in ESCC via negatively regulating DAAM1, suggesting miR-137 as a potent therapeutic candidate for ESCC treatment.

Background: Histone-like proteins are small molecular weight DNA-binding proteins that are widely distributed in prokaryotes. These proteins have multiple functions in cellular structures and processes, including the morphological stability of the nucleoid, DNA compactness, DNA replication, and DNA repair. Deinococcus radiodurans, an extremophilic microorganism, has extraordinary DNA repair capability and encodes an essential histone-like protein, DrHU. Objective: We aim to investigate the phosphorylation regulation role of a histone-like HU protein from Deinococcus radiodurans. Methods: LC-MS/MS analysis was used to determine the phosphorylation site of endogenous DrHU. The predicted structure of DrHU-DNA was obtained from homology modeling (Swissmodel) using Staphylococcus aureus HU-DNA structure (PDB ID: 4QJU) as the starting model. Two types of mutant proteins T37E and T37A were generated to explore their DNA binding affinity. Complemented-knockout strategy was used to generate the ΔDrHU/pk-T37A and ΔDrHU/pk-T37E strains for growth curves and phenotypical analyses. Results and Discussion: The phosphorylation site Thr37, which is present in most bacterial HU proteins, is located at the putative protein-DNA interaction interface of DrHU. Compared to the wild-type protein, one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation (T37E) showed enhanced double-stranded DNA binding but a weakened protective effect against hydroxyl radical cleavage. Complementation of T37E in a DrHU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress. Conclusions: Phosphorylation modulates the DNA-binding capabilities of the histone-like HU protein from D. radiodurans, which contributes to the environmental adaptation of this organism.