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Structural studies of UBXN2A and mortalin interaction and the putative role of silenced UBXN2A in preventing response to chemotherapy.

ABSTRACT: Overexpression of the oncoprotein mortalin in cancer cells and its protein partners enables mortalin to promote multiple oncogenic signaling pathways and effectively antagonize chemotherapy-induced cell death. A UBX-domain-containing protein, UBXN2A, acts as a potential mortalin inhibitor. This current study determines whether UBXN2A effectively binds to and occupies mortalin's binding pocket, resulting in a direct improvement in the tumor's sensitivity to chemotherapy. Molecular modeling of human mortalin's binding pocket and its binding to the SEP domain of UBXN2A followed by yeast two-hybrid and His-tag pull-down assays revealed that three amino acids (PRO442, ILE558, and LYS555) within the substrate-binding domain of mortalin are crucial for UBXN2A binding to mortalin. As revealed by chase experiments in the presence of cycloheximide, overexpression of UBXN2A seems to interfere with the mortalin-CHIP E3 ubiquitin ligase and consequently suppresses the C-terminus of the HSC70-interacting protein (CHIP)-mediated destabilization of p53, resulting in its stabilization in the cytoplasm and upregulation in the nucleus. Overexpression of UBXN2A causes a significant inhibition of cell proliferation and the migration of colon cancer cells. We silenced UBXN2A in the human osteosarcoma U2OS cell line, an enriched mortalin cancer cell, followed by a clinical dosage of the chemotherapeutic agent 5-fluorouracil (5-FU). The UBXN2A knockout U2OS cells revealed that UBXNA is essential for the cytotoxic effect achieved by 5-FU. UBXN2A overexpression markedly increased the apoptotic response of U2OS cells to the 5-FU. In addition, silencing of UBXN2A protein suppresses apoptosis enhanced by UBXN2A overexpression in U2OS. The knowledge gained from this study provides insights into the mechanistic role of UBXN2A as a potent mortalin inhibitor and as a potential chemotherapy sensitizer for clinical application.

SUBMITTER: Sane S

PROVIDER: S-EPMC4786526 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Structural studies of UBXN2A and mortalin interaction and the putative role of silenced UBXN2A in preventing response to chemotherapy.

Sane Sanam S Abdullah Ammara A Nelson Morgan E ME Wang Hongmin H Chauhan Subhash C SC Newton Samuel S SS Rezvani Khosrow K

Cell stress & chaperones 20151204 2

Overexpression of the oncoprotein mortalin in cancer cells and its protein partners enables mortalin to promote multiple oncogenic signaling pathways and effectively antagonize chemotherapy-induced cell death. A UBX-domain-containing protein, UBXN2A, acts as a potential mortalin inhibitor. This current study determines whether UBXN2A effectively binds to and occupies mortalin's binding pocket, resulting in a direct improvement in the tumor's sensitivity to chemotherapy. Molecular modeling of hum ...[more]

PMID: 26634371

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Project description:BackgroundIn human immunodeficiency virus-1 (HIV-1) infection, activation of astrocytes induces imbalance in physiological functions due to perturbed astrocytic functions that unleashes toxicity on neurons. This leads to inflammatory response finally culminating into neurocognitive dysfunction. In neuroAIDS, HIV-1 protein, transactivator of transcription (Tat) is detected in the cerebrospinal fluid of infected patients. Mortalin, a multifunctional protein, has anti-inflammatory role following its activation in various stress conditions. Recent studies demonstrate downregulation of mortalin in neurodegenerative diseases. Here, we explored the mechanisms of mortalin in modulating HIV-1 Tat-mediated neuroinflammation.MethodsExpression of mortalin in autopsy section in normal and diseased individuals were examined using immunohistochemistry. To decipher the role of mortalin in HIV-1 Tat-induced activation, human fetal brain-derived astrocytes were transiently transfected with Tat and mortalin using expression vectors. HIV-1 Tat-mediated damage was analyzed using RT-PCR and western blotting. Modulatory role of mortalin was examined by coexpressing it with Tat, followed by examination of mitochondrial morphodynamics using biochemical assay and confocal and electron microscopy. Extracellular ATP release was monitored using luciferase assay. Neuroinflammation in astrocytes was examined using flow cytometry, dye based study, immunocytochemistry, immunoprecipitation, and western blotting. Indirect neuronal damage was also analyzed.ResultsHIV-1 Tat downregulates the expression of mortalin in astrocytes, and this is corroborated with autopsy sections of HIV-1 patients. We found that overexpression of mortalin with Tat reduced inflammation and also rescued astrocytic-mediated neuronal death. Using bioinformatics, we discovered that binding of mortalin with Tat leads to Tat degradation and rescues the cell from neuroinflammation. Blocking of proteosomal pathway rescued the Tat degradation and revealed the ubiquitination of Tat.ConclusionOverall, our data demonstrated the protective role of mortalin in combating HIV-1 Tat-mediated damage. We also showed that mortalin could degrade Tat through direct binding with HIV-1 Tat. Overexpression of mortalin in the presence of Tat could significantly reduce cytotoxic effects of Tat in astrocytes. Indirect neuronal death was also found to be rescued. Our in vitro findings were validated as we found attenuated expression of mortalin in the autopsy sections of HIV-1 patients.

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Structural studies of UBXN2A and mortalin interaction and the putative role of silenced UBXN2A in preventing response to chemotherapy.

Publications

Structural studies of UBXN2A and mortalin interaction and the putative role of silenced UBXN2A in preventing response to chemotherapy.

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