title: Modeling the in vitro 20S proteasome activity: the effect of PA28-alphabeta and of the sequence and length of polypeptides on the degradation kinetics.
abstract: Proteasomes are fundamental for the degradation of intracellular proteins, having a key role in several important metabolic and signaling pathways, in the cell cycle and in antigen presentation. In vitro proteasomal digestion assays are widely used in molecular biology and immunology. We developed a model, ProteaMAlg (proteasome modeling algorithm) that describes the kinetics of specific protein fragments generated by 20S proteasomes in different conditions, once the substrate cleavage strengths are provided. ProteaMAlg was tested on a variety of data available in the literature as well as on new degradation experiments performed with polypeptides of different sequences and lengths. The comparison between in vitro and in silico experiments was used to quantify the effect on degradation of the sequence and the length of target polypeptides, of the presence of regulatory molecules such as PA28-alphabeta, and of the type of 20S proteasome (constitutive- or immunoproteasome). The model showed that the effect of the PA28 regulatory subunit results in a modification of the gating functions of the proteasome core particle. Immunoproteasome digestion experiments suggested that this form of proteasome, which is involved in generating MHC-class I epitopes, presents modified cleavage and gating activities. Our analysis improves the current understanding of the kinetics of proteasome functioning, and provides a tool to quantify and predict the effect of key parameters during in vitro digestion. ProteaMAlg is publicly available on the web (http://www.proteamalg.com).
authors.affiliation: Interdepartmental Center for Studies on Biophysics, Bioinformatics and Biocomplexity L. Galvani (CIG), University of Bologna, via S. Giacomo 12, 40126 Bologna, Italy. michele.mishto@unibo.it
authors.forename: Michele
authors.initials: M
authors.lastname: Mishto
authors.forename: Fabio
authors.initials: F
authors.lastname: Luciani
authors.forename: Hermann-Georg
authors.initials: HG
authors.lastname: Holzhütter
authors.forename: Elena
authors.initials: E
authors.lastname: Bellavista
authors.forename: Aurelia
authors.initials: A
authors.lastname: Santoro
authors.forename: Kathrin
authors.initials: K
authors.lastname: Textoris-Taube
authors.forename: Claudio
authors.initials: C
authors.lastname: Franceschi
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kloetzel
authors.forename: Alexey
authors.initials: A
authors.lastname: Zaikin
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D006801:Humans; D007700:Kinetics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D009124:Muscle Proteins; D010446:Peptide Fragments; C486207:PSME2 protein, human; D046988:Proteasome Endopeptidase Complex