Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method.
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.
Scanlon
Thomas C
TC
Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, Montreal, Canada.
Gottlieb
Bruce
B
Durcan
Thomas M
TM
Fon
Edward A
EA
Beitel
Lenore K
LK
Trifiro
Mark A
MA
D000074183:ATPases Associated with Diverse Cellular Activities; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis / Q000187:drug effects; D002460:Cell Line; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003374:Coumarins / Q000494:pharmacology; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D003374:Coumarins; D004268:DNA-Binding Proteins; D007976:Leupeptins; D009842:Oligopeptides; D010446:Peptide Fragments; D021122:Protein Subunits; D011506:Proteins; D025801:Ubiquitin; C086985:RAD23A protein, human; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C046582:succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D046988:Proteasome Endopeptidase Complex; D000074183:ATPases Associated with Diverse Cellular Activities; C064757:PSMC3 protein, human; D044764:Ubiquitin-Activating Enzymes; D045643:DNA Repair Enzymes; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde