Assessment of proteasomal cleavage probabilities from kinetic analysis of time-dependent product formation.
Proteasomes are multicatalytic cellular protease complexes that degrade intracellular proteins into smaller peptides. Proteasomal in vitro digests have revealed that the various peptide bonds of a given substrate are cleaved in a highly selective manner. Regarding the key role of proteasomes as the main supplier of antigenic peptides for MHC class I-mediated antigen presentation, it is important to know to what extent these preferences for specific peptide bonds may vary among proteasomes of different cellular origin and of different subunit composition. Here, we quantify such cleavage rates by means of a kinetic proteasome model that relates the time-dependent changes of the amount of any generated peptide to the rates with which this peptide can be either generated from longer precursor peptides or degraded into smaller successor peptides. Numerical values for these rates are estimated by minimizing the distance between simulated and measured time-courses. The proposed method is applied to kinetic data obtained by combining HPLC fractionation and mass spectrometry (MS) to trace the degradation of two model peptides (pp89-25mer and LLO-27mer) by either the constitutive (T2) or immunoproteasome (T2.27). To convert the intensity of the MS signals into the respective peptide amounts, we use two methods leading to similar results: experimental calibration curves and theoretically determined linear scaling functions based on a novel approach using mass conservation rules. Comparison of the cleavage probabilities and procession rates obtained for the two types of proteasomes reveals that the striking differences between the time-dependent peptide profiles can be accounted for mainly by a generally higher turnover rate of the immunoproteasome. For the pp89-25mer, there is no significant change of the cleavage probabilities for any of the ten observed cleavage sites. For the LLO-27mer, there appears to be a significant change in the cleavage probabilities for four of the nine observed cleavage sites when switching from the constitutive to the immunoproteasome.
Peters
Björn
B
Medizinische Fakultät, Charité, Institut für Biochemie, Humboldt Universität Berlin, Monbijoustr. 2, D-10117 Berlin, Germany.
Janek
Katharina
K
Kuckelkorn
Ulrike
U
Holzhütter
Hermann-Georg
HG
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; C071313:LMP-2 protein; D003546:Cysteine Endopeptidases; C077436:LMP7 protein; D046988:Proteasome Endopeptidase Complex