Changes in proteasome structure and function caused by HAMLET in tumor cells.
BACKGROUND
Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death.


RESULTS
HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells.


CONCLUSIONS
The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.
Department of Microbiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.
Lotta
L
Gustafsson
Sonja
S
Aits
Patrik
P
Onnerfjord
Maria
M
Trulsson
Petter
P
Storm
Catharina
C
Svanborg
D016923:Cell Death / Q000502:physiology*; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism*; D007976:Leupeptins / Q000378:metabolism; D008958:Models, Molecular; D019301:Oleic Acid / Q000378:metabolism; D009829:Oleic Acids / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D040081:Protein Array Analysis; D011487:Protein Conformation; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; C506422:HAMLET complex, human; D007976:Leupeptins; D009829:Oleic Acids; D021122:Protein Subunits; D019301:Oleic Acid; D007768:Lactalbumin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde