ABSTRACT
In selective autophagy, receptors are central for cargo selection and delivery. However, it remains yet unclear whether and how multiple autophagy receptors might form complex and function concertedly to control autophagy. Optineurin (OPTN), implicated genetically in glaucoma and amyotrophic lateral sclerosis, was a recently identified autophagy receptor. Here we report that tumor-suppressor HACE1, a ubiquitin ligase, ubiquitylates OPTN and promotes its interaction with p62/SQSTM1 to form the autophagy receptor complex, thus accelerating autophagic flux. Interestingly, the Lys48-linked polyubiquitin chains that HACE1 conjugates onto OPTN might predominantly target OPTN for autophagic degradation. By demonstrating that the HACE1-OPTN axis synergistically suppresses growth and tumorigenicity of lung cancer cells, our findings may open an avenue for developing autophagy-targeted therapeutic intervention into cancer.
Subject(s)
Autophagy , Eye Proteins/metabolism , Lung Neoplasms/enzymology , Transcription Factor TFIIIA/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation , DNA Damage , Eye Proteins/genetics , HEK293 Cells , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Lung Neoplasms/prevention & control , Lysine , Macrophages/enzymology , Membrane Transport Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Oxidative Stress , Protein Binding , RNA Interference , Sequestosome-1 Protein , Signal Transduction , Time Factors , Transcription Factor TFIIIA/genetics , Transfection , Tumor Burden , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in a tumorigenicity assay, iron deprivation suppressed wild-type p53-dependent tumor growth, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but may also help predict and improve outcomes in iron-deprivation-based chemotherapy.
Subject(s)
Iron/metabolism , Tumor Suppressor Protein p53/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Carcinogenesis/metabolism , Cell Line, Tumor , Cytosol/metabolism , Down-Regulation , Heme/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Iron Overload/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Molecular Sequence Data , Sequence Homology, Amino Acid , Signal Transduction , Transcription, Genetic , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
To study its contribution to the assembly of the green plant manganese stabilizing protein (MSP) into photosystem II (PSII), tyrosine residues were specifically acetylated using N-acetylimidazole (NAI). In soluble MSP, three groups of Tyr residues could be differentiated by NAI acetylation: approximately 5 (actually approximately 5.2) Tyr residues could be easily acetylated (superficial), 1-2 Tyr residues could be acetylated when the NAI concentration was sufficiently high (superficially buried), and 1-2 Tyr residues could only be acetylated in the presence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did not affect the reconstitution or oxygen-evolving activities of the MSP, and far-UV circular dichroism (CD) analysis showed that the altered MSP retained most of its native secondary structure. These results suggested that the 5.2 Tyr residues are not absolutely essential to the function of MSP. However, further modification of the 1-2 superficially buried Tyr residues (for a total acetylation of approximately 6.4 Tyr residues) completely abrogated the MSP rebinding and oxygen evolution activities. Finally, at least one tyrosine residue was inaccessible to NAI until MSP was completely unfolded by 8 M urea. Deacetylation of MSP with 6.4 or 8 acetylated Tyr residues with hydroxylamine restored most of the rebinding and oxygen-evolving activities. A prominent red shift in fluorescence spectra of MSP (excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues, and a further shift could be found after all 8 Tyr residues were modified, indicating a great loss of native secondary structure. Far-UV CD revealed that MSP was mostly unfolded when 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues were modified. Fluorescence and far-UV CD studies revealed that loss of MSP rebinding to PSII membranes following NAI modification correlated well with conformational changes in MSP. Together, these results indicate that different tyrosine residues have different contributions to the binding and assembly of MSP into PSII.
Subject(s)
Imidazoles/metabolism , Manganese/chemistry , Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Spinacia oleracea , Tyrosine/chemistry , Acetylation , Amino Acid Sequence , Circular Dichroism , Manganese/metabolism , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Spectrometry, Fluorescence , Structure-Activity Relationship , Thylakoids/chemistry , Thylakoids/metabolism , Tyrosine/metabolismABSTRACT
The extrinsic 33 kD protein of photosystemII(PSII) plays an important role in the stabilizing of manganese cluster and maintaining high oxygen-evolving activity of PSII. In this research, (241)Trp, the only tryptophan in the 33 kD protein, was modified by N-Bromosuccinimide. The pH-dependence of modification suggests that this tryptophan is buried in the hydrophobic interior of the protein. The protein's capability of reconstitution to the PSII ecreased after modification, and no oxygen-evolving activity of PS was recovered after the reconstitution. Results suggest that (241)Trp of the 33 kD protein is essential for the binding of the protein to the PS and the normal oxygen-evolving activity.