Generic Structures of Cytotoxic Liprotides: Nano‐Sized Complexes with Oleic Acid Cores and Shells of Disordered Proteins

Kaspersen, Jørn Døvling; Pedersen, Jannik Nedergaard; Hansted, Jon G.; Nielsen, Søren Achim; Sakthivel, Srinivasan; Wilhelm, K.; Nemashkalova, Ekaterina L.; Permyakov, Sergei E.; Permyakov, Eugene A.; Oliveira, Cristiano L. P.; Morozova‐Roche, Ludmilla A.; Otzen, Daniel E.

doi:10.1002/cbic.201402407

Cited by 39 publications

(75 citation statements)

References 54 publications

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“…Alpha-lactalbumin protein triggers cell death in normal rat intestinal epithelial cells (63) and virus-transformed mouse leukaemia cells (64). These results and HAMLET results showing HAMLET cytotoxicity superior to that of the oleic acid alone (3)(4)(5)15,17,38,44,50,54), indicate that α-lactalbumin protein is also playing role in HAMLET-induced cell death.…”

Section: Oleic Acid Is the Main Active Componentmentioning

confidence: 66%

“…Other studies found that the protein-lipid complex is more cytotoxic than similar amounts of oleic acid or sodium oleate alone (3)(4)(5)15,17,38,44,50,54). It is not yet clear whether these are true differences in cytotoxicity for the different protein carriers of oleate, or whether these different results reflect difficulties in solubilising oleic acid alone for delivery in water-based cellular systems, or are a result of accuracy and precision in measuring the oleate amounts present in the HAMLET-like compounds.…”

Section: Oleic Acid Is the Main Active Componentmentioning

confidence: 97%

“…The amount of oleate that can be added to a partiallyunfolded protein to form a cytotoxic HAMLETlike compound is approximately 1 oleate per 1 kDa molecular weight of protein (5). Adding less oleate during preparation than the maximum amount results in less oleate being complexed with protein and approximately correspondingly less cytotoxicity (higher LD50) (4).…”

Section: Oleic Acid Is the Main Active Componentmentioning

confidence: 99%

“…HAMLET-like compounds characterised to date consist of partially-unfolded protein, usually a milk protein, complexed with oleic acid (1), a similar fatty acid (2,3), or sodium oleate (4,5). The protein is partially unfolded and the available structural evidence suggests that the protein polypeptide, referred to in this review simply as protein, is partially folded around the oleate molecules that are aggregated in the centre of the complex (5,6).…”

Section: Introductionmentioning

confidence: 99%

“…The protein is partially unfolded and the available structural evidence suggests that the protein polypeptide, referred to in this review simply as protein, is partially folded around the oleate molecules that are aggregated in the centre of the complex (5,6). The fatty acid is in the free monomeric form, which may make membranes and membrane proteins particularly sensitive to it (Figure 1), and is not in the triglyceride form of the oleic acid in olive oil.…”

Section: Introductionmentioning

confidence: 99%

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Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components

et al. 2015

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-The HAMLET family of compounds (Human Alpha-lactalbumin Made Lethal to Tumours) was discovered during studies on the properties of human milk, and is a class of protein-lipid complexes having broad spectrum anti-cancer, and some specific anti-bacterial properties. The structure of HAMLETlike compounds consists of an aggregation of partially unfolded protein making up the majority of the compound's mass, with fatty acid molecules bound in the hydrophobic core. This is a novel protein-lipid structure and has only recently been derived by small-angle X-ray scattering analysis. The structure is the basis of a novel cytotoxicity mechanism responsible for anti-cancer activity to all of the around 50 different cancer cell types for which the HAMLET family has been trialled. Multiple cytotoxic mechanisms have been hypothesised for the HAMLET-like compounds, but it is not yet clear which of those are the initiating cytotoxic mechanism(s) and which are subsequent activities triggered by the initiating mechanism(s). In addition to the studies into the structure of these compounds, this review presents the state of knowledge of the anti-cancer aspects of HAMLET-like compounds, the HAMLET-induced cytotoxic activities to cancer and non-cancer cells, and the several prospective cell membrane and intracellular targets of the HAMLET family. The emerging picture is that HAMLET-like compounds initiate their cytotoxic effects on what may be a cancer-specific target in the cell membrane that has yet to be identified.

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Section: Oleic Acid Is the Main Active Componentmentioning

confidence: 66%

Section: Oleic Acid Is the Main Active Componentmentioning

confidence: 97%

Section: Oleic Acid Is the Main Active Componentmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components

et al. 2015

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Role of Charge and Hydrophobicity in Liprotide Formation: A Molecular Dynamics Study with Experimental Constraints

et al. 2018

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Bovine α-lactalbumin (aLA) and oleate (OA) form a complex that has been intensively studied for its tumoricidal activity. Small-angle X-ray scattering (SAXS) has revealed that this complex consists of a lipid core surrounded by partially unfolded protein. We call this type of complex a liprotide. Little is known of the molecular interactions between OA and aLA, and no technique has so far provided any high-resolution structure of a liprotide. Here we have used coarse-grained (CG) molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC) and SAXS to investigate the interactions between aLA and OA during the process of liprotide formation. With ITC we found that the strongest enthalpic interactions occurred at a molar ratio of 12.0±1.4:1 OA/aLA. Liprotides formed between OA and aLA at several OA/aLA ratios in silico were stable both in CG and in all-atom simulations. From the simulated structures we calculated SAXS spectra that show good agreement with experimentally measured patterns of matching liprotides. The simulations showed that aLA assumes a molten globular (MG) state, exposing several hydrophobic patches involved in interactions with OA. Initial binding of aLA to OA occurs in an area of aLA in which a high amount of positive charge is located, and only later do hydrophobic interactions become important. The results reveal how unfolding of aLA to expose hydrophobic residues is important for complex formation between aLA and OA. Our findings suggest a general mechanism for liprotide formation and might explain the ability of a large number of proteins to form liprotides with OA.

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Liprotides assist in folding of outer membrane proteins

Pedersen

Otzen

2017

Protein Science

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Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α-lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α-lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non-ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8-9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding-inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins.

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Generic Structures of Cytotoxic Liprotides: Nano‐Sized Complexes with Oleic Acid Cores and Shells of Disordered Proteins

Cited by 39 publications

References 54 publications

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components

Role of Charge and Hydrophobicity in Liprotide Formation: A Molecular Dynamics Study with Experimental Constraints

Liprotides assist in folding of outer membrane proteins

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