title: The proteosomal degradation of fusion proteins cannot be predicted from the proteosome susceptibility of their individual components.
abstract: It is assumed that the proteosome-processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome-degradable proteins does not necessarily result in a proteosome-degradable chimera. Conversely, fusion of proteosome-resistant proteins may result in a proteosome-degradable composite. We previously demonstrated that conserved influenza proteins can be unified into a single fusion antigen that is protective, and that vaccination with combinations of proteosome-resistant and proteosome-degradable antigens resulted in an augmented T-cell response. In the present study we constructed proteosome-degradable mutants of conserved influenza proteins NP, M1, NS1, and M2. These were then fused into multipartite proteins in different positions. The stability and degradation profiles of these fusion constructs were demonstrated to depend on the relative position of the individual proteins within the chimeric molecule. Combining unstable sequences of either NP and M1 or NS1 and M2 resulted in either rapidly proteosome degraded or proteosome-resistant bipartite fusion mutants. However, further unification of the proteosome-degradable forms into a single four-partite fusion molecule resulted in relatively stable chimeric proteins. Conversely, the addition of proteosome-resistant wild-type M2 to proteosome-resistant NP-M1-NS1 fusion protein lead to the decreased stability of the resulting four-partite multigene products, which in one case was clearly proteosome dependent. Additionally, a highly destabilized form of M1 failed to destabilize the wild-type NP. Collectively, we did not observe any additive effect leading to proteosomal degradation/nondegradation of a multigene construct.
authors.affiliation: Cure Lab, Inc., Canton, Massachusetts 02021, USA. ilyinskii@curelab.com
authors.forename: Petr O
authors.initials: PO
authors.lastname: Ilyinskii
authors.forename: Anatoli B
authors.initials: AB
authors.lastname: Meriin
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Gabai
authors.forename: Evgeny V
authors.initials: EV
authors.lastname: Usachev
authors.forename: Alexei G
authors.initials: AG
authors.lastname: Prilipov
authors.forename: Galini
authors.initials: G
authors.lastname: Thoidis
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Shneider
mesh_terms: D006868:Hydrolysis; D009975:Orthomyxoviridae / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D046988:Proteasome Endopeptidase Complex