Evidence that melatonin downregulates Nedd4-1 E3 ligase and its role in cellular survival.

Indolamine melatonin structurally resembles non-covalent proteasome inhibitors; however, the role of ubiquitin proteasome system (UPS) in neuronal survival and how melatonin carries out UPS inhibition remain largely unknown. With the use of melatonin treated cells, we evaluated the expression of Nedd4-1, an E3 ligase, how melatonin regulates its activity and its relationship with neuronal survival. Nedd4-1 was upregulated in the hypoxic condition in both control and Nedd4-1 overexpressed cells and melatonin treatment reversed its expression in both normoxic and hypoxic conditions, which was associated with increased cellular survival. Melatonin had no effect on the expression of Nedd4-1 at mRNA level. However, when melatonin was administered along with protein synthesis inhibitor cycloheximide, protein level of Nedd4-1 was further reduced, indicating that melatonin possibly downregulates Nedd4-1 after its synthesis. Notably, co-immunoprecipitation analyses followed by Liquid chromatography-Mass Spectrometry (LC-MS/MS) revealed that melatonin may dissociate ribosomal proteins, such as RS19, RL23A, and nucleophosmin from Nedd4-1, while 40S ribosomal protein S7 and 60S ribosomal protein L35 came into contact with Nedd4-1 upon melatonin treatment. By using IPA analyses, we obtained further data indicated novel target molecules of melatonin in hypoxic conditions, including OTOF, SF3B2, IPO5, ST13, FGFR3, Mx1/Mx2, playing roles in RNA splicing and trafficking, growth factor and interferon signaling. Here, we described a new insight into the role of melatonin in UPS functioning by proposing a molecular mechanism through which melatonin regulates Nedd4-1.

Extracellular matrix collagen biomarker levels in patients who underwent pulmonary endarterectomy.

OBJECTIVES: The role of extracellular matrix collagen biomarkers in chronic thromboembolic pulmonary hypertension (CTEPH) is not well known. Our goal was to investigate the matrix metalloproteinase (MMP)-2 and -9 protein levels in patients with CTETH. METHODS: This is a prospective, cross-sectional study. Patients with CTETH who underwent pulmonary endarterectomy comprise group 1, and the control group included patients who underwent lung surgery without pulmonary hypertension (group 2) between March 2020 and March 2021. In addition to serum levels of MMP-9, the pulmonary endarterectomy and control pulmonary artery tissue samples were measured by the enzyme-linked immunosorbent assay  4pl, cubic, quadratic and Western blot techniques. Levels of MMP-2, which consist of pro MMP-2/ß-actin and active MMP-2/ß-actin and MMP-9/ß-actin, were measured only in the tissue samples. RESULTS: Forty-eight patients were enrolled consecutively in group 1 (n: 24) and group 2 (n: 24). The serum concentrations of MMP-9 were similar in both groups. Similarly, a comparison of tissue sample levels of pro MMP-2/ß-actin (P = 0.496) and active MMP-2/ß-actin (P = 0.216) showed no significant difference between the groups. The tissue samples from patients with CTETH had significantly lower amounts of MMP-9/ß-actin compared to the control group (P = 0.001). CONCLUSIONS: This study indicates that serum levels of extracellular matrix collagen biomarkers were similar in patients with CTETH who were candidates for surgery and in patients who had non-pulmonary hypertension who underwent lung surgery. Differences in levels of MMP-9/ß-actin in tissue samples may play a role in pulmonary vascular remodelling in operable patients.