title: Functional 20S proteasomes in mature human red blood cells.
abstract: The purpose of the present study was to investigate whether functional 20S and/or 26S proteasomes are present within mature human red blood cells (RBCs; depleted of reticulocytes and leukocytes). Double-immunofluorescence confocal microscopy showed the presence of immunoreactive 20S and 19S proteasomal subunit proteins and their partial co-localization within mature RBCs. Proteasomes isolated from mature RBCs displayed 20S activity in vitro; atomic-force and transmission electron microscopy of isolated proteasomes revealed abundant 20S core particles and very few 26S particles. A two-dimensional differential in-gel electrophoresis (2D-DIGE) approach was used to determine if proteasome-dependent protein degradation occurs within mature RBCs. Twenty-eight proteins were identified with altered protein content in response to lactacystin. Seven cytosolic proteins showed an increase and 16 showed a decrease; five membrane proteins showed a decrease. We conclude that the proteins showing increased abundance are either primary or secondary targets of the 20S proteasome and that putatively degraded proteins are secondary targets. Therefore, functional 20S proteasomes exist within mature RBCs. Our study did not detect 26S proteasome activity using the 2D-DIGE approach.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, 13210, USA.
authors.forename: Sudha
authors.initials: S
authors.lastname: Neelam
authors.forename: David G
authors.initials: DG
authors.lastname: Kakhniashvili
authors.forename: Stephan
authors.initials: S
authors.lastname: Wilkens
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Levene
authors.forename: Steven R
authors.initials: SR
authors.lastname: Goodman
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D001798:Blood Proteins / Q000378:metabolism; D002454:Cell Differentiation; D015180:Electrophoresis, Gel, Two-Dimensional; D004912:Erythrocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism*; D061988:Proteasome Inhibitors; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology
chemical_list: D001798:Blood Proteins; D008565:Membrane Proteins; D061988:Proteasome Inhibitors; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine