| 1. |
- Permyakov, Sergei E, et al.
(author)
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A novel method for preparation of HAMLET-like protein complexes
- 2011
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In: Biochimie. - : Elsevier BV. - 1638-6183 .- 0300-9084. ; 93:9, s. 501-1495
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Journal article (peer-reviewed)abstract
- Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca(2+) transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.
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| 2. |
- Permyakov, Sergei E, et al.
(author)
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Oleic acid is a key cytotoxic component of HAMLET-like complexes
- 2012
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In: Biological Chemistry. - 1437-4315. ; 393:1-2, s. 85-92
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Journal article (peer-reviewed)abstract
- HAMLET is a complex of α-lactalbumin (α-LA) with oleic acid (OA) that selectively kills tumor cells and Streptococcus pneumoniae. To assess the contribution of the proteinaceous component to cytotoxicity of HAMLET, OA complexes with proteins structurally and functionally distinct from α-LA were prepared. Similar to HAMLET, the OA complexes with bovine β-lactoglobulin (bLG) and pike parvalbumin (pPA) (bLG-OA-45 and pPA-OA-45, respectively) induced S. pneumoniae D39 cell death. The activation mechanisms of S. pneumoniae death for these complexes were analogous to those for HAMLET, and the cytotoxicity of the complexes increased with OA content in the preparations. The half-maximal inhibitory concentration for HEp-2 cells linearly decreased with rise in OA content in the preparations, and OA concentration in the preparations causing HEp-2 cell death was close to the cytotoxicity of OA alone. Hence, the cytotoxic action of these complexes against HEp-2 cells is induced mostly by OA. Thermal stabilization of bLG upon association with OA implies that cytotoxicity of bLG-OA-45 complex cannot be ascribed to molten globule-like conformation of the protein component. Overall, the proteinaceous component of HAMLET-like complexes studied is not a prerequisite for their activity; the cytotoxicity of these complexes is mostly due to the action of OA.
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| 3. |
- Rath, Emma M, et al.
(author)
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Small-angle X-ray scattering of BAMLET at pH 12 : a complex of α-lactalbumin and oleic acid
- 2014
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In: Proteins. - : Wiley. - 0887-3585. ; 82:7, s. 8-1400
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Journal article (peer-reviewed)abstract
- BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-like complexes, a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X-ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo-α-lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo-α-lactalbumin was performed and the results suggest that α-lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non-specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure.
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| 4. |
- Håkansson, Anders P, et al.
(author)
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Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex
- 2011
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:3
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Journal article (peer-reviewed)abstract
- Background: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.
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| 5. |
- Logsdon, Lauren K, et al.
(author)
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Streptolysin O inhibits clathrin-dependent internalization of group A Streptococcus
- 2011
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In: mBio. - 2161-2129. ; 2:1, s. 10-00332
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Journal article (peer-reviewed)abstract
- Group A Streptococcus (GAS) can be internalized by epithelial cells, including keratinocytes from human skin or pharyngeal epithelium. Internalization of GAS by epithelial cells has been postulated both to play a role in host defense and to provide a sanctuary site for GAS survival. The cholesterol-binding cytolysin streptolysin O (SLO) appears to enhance virulence in part by inhibiting GAS internalization by human keratinocytes and by disrupting the lysosomal degradation of internalized GAS. We now report that low-level production of SLO by an inducible expression system reduced GAS internalization by keratinocytes. Induced SLO expression also prevented lysosomal colocalization with intracellular bacteria and acidification of GAS-containing vacuoles. Exogenous recombinant SLO mimicked the inhibitory effect of SLO secretion on GAS entry but not that on colocalization with the lysosomal marker LAMP-1, implying that disruption of lysosomal degradation requires intracellular secretion of SLO. The internalization of SLO-negative GAS was blocked by the depletion of host cell cholesterol and by the inhibition or knocking down of the expression of clathrin or dynamin. SLO also inhibited the cellular uptake of other cargos that are internalized by clathrin-mediated uptake or by macropinocytosis. We conclude that SLO interferes with the internalization of GAS through local perturbation of the keratinocyte cell membrane and disruption of a clathrin-dependent uptake pathway.
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