Mapping and structural dissection of human 20 S proteasome using proteomic approaches.
The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).
Claverol
Stephane
S
Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse, France.
Burlet-Schiltz
Odile
O
Girbal-Neuhauser
Elisabeth
E
Gairin
Jean Edouard
JE
Monsarrat
Bernard
B
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009097:Multienzyme Complexes; D021122:Protein Subunits; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex