adenosine-5--o-(3-thiotriphosphate) and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.

Topics: Adenosine Triphosphate; ATPases Associated with Diverse Cellular Activities; Binding Sites; Catalysis; Cell Line; Chromatography, Affinity; Coumarins; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Enzyme Stability; Humans; Leupeptins; Oligopeptides; Peptide Fragments; Proteasome Endopeptidase Complex; Protein Binding; Protein Subunits; Proteins; Tandem Mass Spectrometry; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases

The AF-2 helix of nuclear receptors is essential for ligand-activated transcription, and it may function to couple the receptor to transcriptional coactivator proteins. This domain also contacts components of the proteasome machinery, suggesting that nuclear receptors may be targets for proteasome-mediated proteolysis. In the present study, we demonstrate that mSUG1 (P45), a component of the 26S proteasome, interacts in a 1,25-(OH)2D3-dependent manner with the AF-2 domain of the vitamin D receptor (VDR). Furthermore, treatment of ROS 17/2.8 osteosarcoma cells with the proteasome inhibitors MG132 or beta-lactone increased steady-state levels of the VDR protein. In the presence cycloheximide (10 microg/ml), the liganded VDR protein was degraded with a half-life of approximately 8 h, and this rate of degradation was completely blocked by 0.05 mM MG132. The role of SUG1 -VDR interaction in this process was investigated in transient expression studies. Overexpression of wild-type mSUG1 in ROS17/2.8 cells generated a novel proteolytic VDR fragment of approximately 50 kDa, and its production was blocked by proteasome inhibitors or by a nonhydrolyzable ATP analog. Parallel studies with SUG1 (K196H), a mutant that does not interact with the VDR, did not produce the 50 kDa VDR fragment. Functionally, expression of SUG1 in a VDR-responsive reporter gene assay resulted in a profound inhibition of 1,25-(OH)2D3-activated transcription, while expression of SUG1 (K196H) had no significant effect in this system. These data show that the AF-2 domain of VDR interacts with SUG1 in a 1,25-(OH)2D3-dependent fashion and that this interaction may target VDR to proteasome-mediated degradation as a means to downregulate the 1,25-(OH)2D3-activated transcriptional response.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphate; Animals; ATPases Associated with Diverse Cellular Activities; Calcitriol; Carrier Proteins; Cloning, Molecular; Cycloheximide; Cysteine Endopeptidases; Gene Expression Regulation; Half-Life; Intracellular Signaling Peptides and Proteins; Lactones; Leupeptins; Ligands; LIM Domain Proteins; Mice; Multienzyme Complexes; Osteosarcoma; Peptide Fragments; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Structure, Secondary; Receptors, Calcitriol; Transcription Factors; Transfection; Tumor Cells, Cultured