2. Academic Validation
  3. Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda

Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda

  • Biochim Biophys Acta Proteins Proteom. 2019 Sep;1867(9):840-853. doi: 10.1016/j.bbapap.2019.06.010.
Oksana I Kravchuk 1,  Yulia V Lyupina 1,  Pavel A Erokhov 1,  Alexander D Finoshin 1,  Kim I Adameyko 1,  Maryia Yu Mishyna 2,  Andrey V Moiseenko 2,  Olga S Sokolova 2,  Olga V Orlova 3,  Svetlana N Beljelarskaya 3,  Marina V Serebryakova 4,  Maria I Indeykina 5,  Anna E Bugrova 5,  Alexey S Kononikhin 6,  Victor S Mikhailov 7
Affiliations

Affiliations

  • 1 N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova str., Moscow 119334, Russia.
  • 2 M.V. Lomonosov Moscow State University, Faculty of Biology, 1-12 Leninskie Gory, Moscow 119991, Russia.
  • 3 V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova str., Moscow 119334, Russia.
  • 4 A.N. Belozersky Institute of Physico-Chemical Biology MSU, 1c40 Leniniskie Gory, Moscow 119234, Russia.
  • 5 N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygina str., Moscow 119334, Russia.
  • 6 N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygina str., Moscow 119334, Russia; Skolkovo Institute of Science and Technology, 3 Ulitsa Nobelya, Moscow region, Skolkovo 121205, Russia.
  • 7 N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova str., Moscow 119334, Russia. Electronic address: mikhailov48@mail.ru.
PMID: 31228587 DOI: 10.1016/j.bbapap.2019.06.010
Abstract

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct Proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S Proteasome subunits in Sf9 cells was determined by a search of databases and by Sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S Proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.

Keywords

20S proteasome; 20S proteasome subunits; Insects; Lepidoptera; Proteasome activators; Spodoptera frugiperda.

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