Changes in Proteasome Structure and Function Caused by HAMLET in Tumor Cells

Abstract

BackgroundProteasomes 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.Methodology/Principal FindingsHAMLET 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 α-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (β1 and β5) and structural subunits (α2, α3, α6 and β3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells.Conclusions/SignificanceThe 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.

The cytotoxicity of fatty acid/α‐lactalbumin complexes depends on the amount and type of fatty acid

“…HAMLET has also been shown to affect mitochondria [31] and induce apoptosis-like cell death with caspase activation, PS externalization and chromatin condensation irrespective of the p53 and bcl-2 status of the cells [32] and to cause changes in proteasome structure and function [33]. In addition, it has been shown by Aits et al that HAMLET induces autophagy [34], while Rammer et al see activation of the lysosomal death pathway in response to BAMLET treatment, but that the cytotoxicity of BAMLET does not depend on autophagy [18].…”

Examination of the Activity of Camel Milk Casein against Hepatitis C Virus (Genotype-4a) and Its Apoptotic Potential in Hepatoma and HeLa Cell Lines

“…The present results are in agreement with those of Svensson et al (26) who reported that upon unfolding, human and bovine α-lactalbumin can form a tumoricidal complex with oleic acid called human alpha-lactalbumin, which is lethal for tumor cells (HAMLET). This complex induces differential and significant apoptosis in several cancer lines (14,27,28). Camel casein was not able to block or neutralize HCV cell entry either by direct or indirect interaction.…”

The Alpha-Lactalbumin/Oleic Acid Complex and Its Cytotoxic Activity

“…In order to clarify the subcellular localization of HAMLET and the interactions of the complex with different tumor cell compartments, researchers have used biotinylated [5,66], I 125 radioactive labeled [66,67] and Alexa Fluor 568-labeled HAMLET complexes in confocal microscopy and subcellular fractionation experiments [67][68][69]. Site-specific labeling of HAMLET and HAMLET-like complexes with aminooxy-Alexa Fluor 488 or biotin molecules has also been used [52].…”

“…In addition, it has been observed that -LA is able to rapidly insert itself into the lipid bilayer at pH 2 [75]. However, native human -LA is only inefficiently bound to and internalized by tumor cells, and does not translocate to the nucleus, nor influence tumor cell viability [3,5,68,69,76]. Even partial unfolding of human -LA has been shown to be insufficient for increased protein internalization into tumor cells [38].…”

“…HAMLET has been shown to interact directly with 20S proteasome subunits in vitro as well as in vivo, resulting in structural modifications and partial inhibition of 20S proteasome activity, possibly leading to accumulation of incorrectly folded proteins contributing to cell death [69]. Moreover, HAMLET was found by N-terminal sequencing and MALDI-TOF-MS to bind intact ribosomes, as well as individual ribosomal proteins L4, L6, L15, L13a, L30, L35a, S12 and L21 [2].…”