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Catharina Svanborg - One of the best experts on this subject based on the ideXlab platform.
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beta sheet specific interactions with heat shock proteins define a mechanism of delayed tumor cell death in response to hamlet
Journal of Molecular Biology, 2019Co-Authors: Aftab Nadeem, Tuan Hiep Tran, Sanchari Paul, Victoria Granqvist, Nadege Despretz, Catharina SvanborgAbstract:Abstract As chaperones, heat shock proteins (HSPs) protect host cells against misfolded proteins that constitute a by-product of protein synthesis. Certain HSPs are also expressed on the surface of tumor cells, possibly to scavenge extracellular unfolded protein ligands and prevent them from becoming cytotoxic. HAMLET—a complex of partially unfolded Alpha-Lactalbumin and oleic acid—is relying on its N-terminal alpha-helical domain to perturb tumor cell membranes, and the cells die as a consequence of this interaction. Here we show that in parallel, cell surface HSPs bind the beta-sheet domain of Alpha-Lactalbumin and activate a temporarily protective loop, involving vesicular uptake and lysosomal accumulation. Later, HAMLET destroys lysosomal membrane integrity, and HAMLET release kills the remaining tumor cells. HSPs were identified as HAMLET targets in a proteomic screen and Hsp70-specific antibodies or shRNAs inhibited HAMLET uptake by tumor cells, which showed increased Hsp70 surface expression compared to differentiated cells. The results suggest that HAMLET engages tumor cells by two parallel recognition mechanisms, defined by alpha-helical- or beta-sheet domains of Alpha-Lactalbumin and resulting in an immediate death response, or a delay due to transient accumulation of the complex in the lysosomes. This dual response pattern was conserved among tumor cells but not seen in normal, differentiated cells. By two different mechanisms, HAMLET thus achieves a remarkably efficient elimination of tumor cells.
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α lactalbumin engineered to be nonnative and inactive kills tumor cells when in complex with oleic acid a new biological function resulting from partial unfolding
Journal of Molecular Biology, 2009Co-Authors: Jenny Petterssonkastberg, Ann-kristin Mossberg, Catharina Svanborg, Maria Trulsson, Yeon Joong Yong, Soyoung Min, Yoongho Lim, John E ObrienAbstract:HAMLET (human Alpha-Lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded Alpha-Lactalbumin on the stationary phase in situ. Because native Alpha-Lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human Alpha-Lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify Alpha-Lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.
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bladder cancers respond to intravesical instillation of hamlet human alpha lactalbumin made lethal to tumor cells
International Journal of Cancer, 2007Co-Authors: Ann-kristin Mossberg, Lotta Gustafsson, Bjorn Wullt, Wiking Mansson, Eva Ljunggren, Catharina SvanborgAbstract:We studied if bladder cancers respond to HAMLET (human Alpha-Lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native Alpha-Lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers.
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alpha lactalbumin species variation hamlet formation and tumor cell death
Biochemical and Biophysical Research Communications, 2006Co-Authors: Jenny Pettersson, Ann-kristin Mossberg, Catharina SvanborgAbstract:HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo α-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated α-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by α-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine α-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of α-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.
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stability of hamlet a kinetically trapped alpha lactalbumin oleic acid complex
Protein Science, 2005Co-Authors: Jonas Fast, Ann-kristin Mossberg, Catharina Svanborg, Sara LinseAbstract:The stability toward thermal and urea denaturation was measured for HAMLET (human α-lactalbumin made lethal to tumor cells) and α-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than α-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo α-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than α-lactalbumin, although it is a complex of α-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
Ann-kristin Mossberg - One of the best experts on this subject based on the ideXlab platform.
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α lactalbumin engineered to be nonnative and inactive kills tumor cells when in complex with oleic acid a new biological function resulting from partial unfolding
Journal of Molecular Biology, 2009Co-Authors: Jenny Petterssonkastberg, Ann-kristin Mossberg, Catharina Svanborg, Maria Trulsson, Yeon Joong Yong, Soyoung Min, Yoongho Lim, John E ObrienAbstract:HAMLET (human Alpha-Lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded Alpha-Lactalbumin on the stationary phase in situ. Because native Alpha-Lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human Alpha-Lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify Alpha-Lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.
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bladder cancers respond to intravesical instillation of hamlet human alpha lactalbumin made lethal to tumor cells
International Journal of Cancer, 2007Co-Authors: Ann-kristin Mossberg, Lotta Gustafsson, Bjorn Wullt, Wiking Mansson, Eva Ljunggren, Catharina SvanborgAbstract:We studied if bladder cancers respond to HAMLET (human Alpha-Lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native Alpha-Lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers.
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alpha lactalbumin species variation hamlet formation and tumor cell death
Biochemical and Biophysical Research Communications, 2006Co-Authors: Jenny Pettersson, Ann-kristin Mossberg, Catharina SvanborgAbstract:HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo α-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated α-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by α-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine α-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of α-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.
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stability of hamlet a kinetically trapped alpha lactalbumin oleic acid complex
Protein Science, 2005Co-Authors: Jonas Fast, Ann-kristin Mossberg, Catharina Svanborg, Sara LinseAbstract:The stability toward thermal and urea denaturation was measured for HAMLET (human α-lactalbumin made lethal to tumor cells) and α-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than α-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo α-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than α-lactalbumin, although it is a complex of α-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
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treatment of skin papillomas with topical alpha lactalbumin oleic acid
The New England Journal of Medicine, 2004Co-Authors: Lotta Gustafsson, Ann-kristin Mossberg, Irene Leijonhufvud, Annika Aronsson, Catharina SvanborgAbstract:background We studied the effect on skin papillomas of topical application of a complex of a -lactalbumin and oleic acid (often referred to as human a -lactalbumin made lethal to tumor cells [HAMLET]) to establish proof of the principle that a -lactalbumin–oleic acid kills transformed cells but not healthy, differentiated cells. methods Forty patients with cutaneous papillomas that were resistant to conventional treatment were enrolled in a randomized, placebo-controlled, double-blind study, in which a -lactalbumin–oleic acid or saline placebo was applied daily for three weeks and the change in the volume of each lesion was recorded. After this first phase of the study, 34 patients participated in the second phase, an open-label trial of a three-week course of a -lactalbumin–oleic acid. Approximately two years after the end of the open-label phase of the study, 38 of the original 40 patients were examined, and long-term followup data were obtained. results In the first phase of the study, the lesion volume was reduced by 75 percent or more in all 20 patients in the a -lactalbumin–oleic acid group, and in 88 of 92 papillomas; in the placebo group, a similar effect was evident in only 3 of 20 patients (15 of 74 papillomas) (P<0.001). After the patients in the initial placebo group had been treated with a -lactalbumin–oleic acid in the second phase of the study, a median reduction of 82 percent in lesion volume was observed. At follow-up two years after the end of the second phase, all lesions had completely resolved in 83 percent of the patients treated with a -lactalbumin–oleic acid, and the time to resolution was shorter in the group originally assigned to receive a -lactalbumin–oleic acid than among patients originally in the placebo group (2.4 vs. 9.9 months; P<0.01). No adverse reactions were reported, and there was no difference in the outcomes of treatment between immunocompetent and immunosuppressed patients. conclusions Treatment with topical a -lactalbumin–oleic acid has a beneficial and lasting effect on skin papillomas.
Peter S Kim - One of the best experts on this subject based on the ideXlab platform.
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a specific hydrophobic core in the alpha lactalbumin molten globule
Journal of Molecular Biology, 1998Co-Authors: Peter S KimAbstract:Molten globules are partially structured protein folding intermediates that adopt a native-like overall backbone topology in the absence of extensive detectable tertiary interactions. It is important to determine the extent of specific tertiary structure present in molten globules and to understand the role of specific side-chain packing in stabilizing and specifying molten-globule structure. Previous studies indicate that a small degree of specific side-chain packing stabilizes the structures of the cytochrome c, apomyoglobin, and staphylococcal nuclease molten globules. Here we investigate the extent of specific side-chain packing in the molten globule of α-lactalbumin (α-LA), a highly fluctuating, non-cooperatively formed molten globule. By analyzing a set of point mutations in the helical domain of α-LA, we have identified a stabilizing hydrophobic core. Moreover, this core corresponds to a previously identified structural subdomain and likely contains some native-like packing interactions. Our results suggest that native-like packing of core amino acids helps stabilize molten globules and that some specific interactions can exist in even highly dynamic, fluctuating species.
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bipartite structure of the alpha lactalbumin molten globule
Nature Structural & Molecular Biology, 1995Co-Authors: Zhengyu Peng, Peter S KimAbstract:Molten globules are thought to be general intermediates in protein folding. Apparently conflicting studies have failed to clarify whether one of the best characterized molten globules, that of Alpha-Lactalbumin, resembles an expanded native-like protein or a nonspecific collapsed polypeptide. Here we show that the molten globule properties of Alpha-Lactalbumin are largely confined to one of its two domains. The alpha-helical domain forms a helical structure with a native-like tertiary fold, while the beta-sheet domain is largely unstructured. Molten globules thus possess a native-like backbone topology, but this topology does not necessarily encompass the entire polypeptide chain. Our studies indicate that molten globules provide an approximate solution to, and considerable simplification of the protein folding problem.
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local structural preferences in the alpha lactalbumin molten globule
Biochemistry, 1995Co-Authors: Zhengyu Peng, Peter S KimAbstract:Molten globules have been proposed to be general intermediates in protein folding. Despite numerous studies, a detailed description of the structure of a molten globule remains elusive. Recently, we showed that the molten globule formed by the helical domain of Alpha-Lactalbumin (alpha-LA) has a native-like backbone topology. Here we probe local structural preferences in the helical domain of the alpha-LA molten globule by analyzing a set of native and nonnative single disulfide bond variants using a combination of circular dichroism spectroscopy and determination of the equilibrium constant for disulfide bond formation. We find that the region surrounding the 28-111 disulfide bond has a high preference to adopt a native-like structure. Formation of other native or nonnative disulfide bonds is significantly less favorable. Our results suggest that molten globules contain regions with varying degrees of specificity for native-like structure and that the core region surrounding the 28-111 disulfide bond plays an important role in alpha-LA folding by stabilizing the molten globule intermediate.
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a protein dissection study of a molten globule
Biochemistry, 1994Co-Authors: Zhengyu Peng, Peter S KimAbstract:Proteins have many distinct tertiary folds (Richardson, J. S. (1981) Adv. Prot. Chem. 34, 167-339). The term tertiary fold refers to the spatial organization of secondary structure elements (alpha-helices and beta-strands). It is not known when, in the process of protein folding, a native tertiary fold emerges. Here, we show that the helical domain of human Alpha-Lactalbumin, in isolation, forms a molten globule with the same overall tertiary fold as that found in intact Alpha-Lactalbumin. Formation of this native-like fold does not require extensive, specific side-chain packing. Our results suggest that much of the information transfer from one-dimension to three-dimensions has occurred at the molten globule stage of protein folding.
Sara Linse - One of the best experts on this subject based on the ideXlab platform.
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stability of hamlet a kinetically trapped alpha lactalbumin oleic acid complex
Protein Science, 2005Co-Authors: Jonas Fast, Ann-kristin Mossberg, Catharina Svanborg, Sara LinseAbstract:The stability toward thermal and urea denaturation was measured for HAMLET (human α-lactalbumin made lethal to tumor cells) and α-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than α-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo α-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than α-lactalbumin, although it is a complex of α-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
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alpha lactalbumin unfolding is not sufficient to cause apoptosis but is required for the conversion to hamlet human alpha lactalbumin made lethal to tumor cells
Protein Science, 2003Co-Authors: Malin Svensson, Ann-kristin Mossberg, Sara Linse, Lotta Gustafsson, Lawrence J Berliner, Jonas Fast, Caroline Duringer, Oskar Hallgren, Charles L Brooks, Catharina SvanborgAbstract:HAMLET (human α-lactalbumin made lethal to tumor cells) is a complex of human α-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from α-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of α-lactalbumin is sufficient to induce cell death. We used the bovine α-lactalbumin Ca2+ site mutant D87A, which is unable to bind Ca2+, and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine α-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca2+site, as HAMLET maintained a high affinity for Ca2+ but D87A-BAMLET was active with no Ca2+ bound. We conclude that partial unfolding of α-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+-binding site is not required for conversion of α-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca2+site.
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Conversion of Alpha-Lactalbumin to a protein inducing apoptosis.
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Malin Svensson, Ann-kristin Mossberg, Sara Linse, Anders Håkansson, Catharina SvanborgAbstract:In this study α-lactalbumin was converted from the regular, native state to a folding variant with altered biological function. The folding variant was shown to induce apoptosis in tumor cells and immature cells, but healthy cells were resistant to this effect. Conversion to HAMLET (human α-lactalbumin made lethal to tumor cells) required partial unfolding of the protein and a specific fatty acid, C18:1, as a necessary cofactor. Conversion was achieved with α-lactalbumin derived from human milk whey and with recombinant protein expressed in Escherichia coli. We thus have identified the folding change and the fatty acid as two key elements that define HAMLET, the apoptosis-inducing functional state of α-lactalbumin. Although the environment in the mammary gland favors the native conformation of α-lactalbumin that serves as a specifier in the lactose synthase complex, the conditions under which HAMLET was formed resemble those in the stomach of the nursing child. Low pH is known to release Ca2+ from the high-affinity Ca2+-binding site and to activate lipases that hydrolyze free fatty acids from milk triglycerides. We propose that this single amino acid polypeptide chain may perform vastly different biological functions depending on its folding state and the in vivo environment. It may be speculated that molecules like HAMLET can aid in lowering the incidence of cancer in breast-fed children by purging of tumor cells from the gut of the neonate.
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a folding variant of alpha lactalbumin with bactericidal activity against streptococcus pneumoniae
Molecular Microbiology, 2000Co-Authors: Anders Håkansson, Malin Svensson, Ann-kristin Mossberg, Sara Linse, Bo Lonnerdal, Hemant Sabharwal, Irene Lazou, Catharina SvanborgAbstract:This study describes an Alpha-Lactalbumin folding variant from human milk with bactericidal activity against antibiotic-resistant and -susceptible strains of Streptococcus pneumoniae. The active complex precipitated with the casein fraction at pH 4.6 and was purified from casein by a combination of anion exchange and gel chromatography. Unlike other casein components, the active complex was retained on the ion-exchange matrix and eluted only with high salt. The eluted fraction showed N-terminal and mass spectrometric identity with human milk Alpha-Lactalbumin, but native Alpha-Lactalbumin had no bactericidal effect. Spectroscopic analysis demonstrated that the active form of the molecule was in a different folding state, with secondary structure identical to Alpha-Lactalbumin from human milk whey, but fluctuating tertiary structure. Native Alpha-Lactalbumin could be converted to the active bactericidal form by ion-exchange chromatography in the presence of a cofactor from human milk casein, characterized as a C18:1 fatty acid. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram-negative and other Gram-positive bacteria were resistant. The folding variant of Alpha-Lactalbumin is a new example of naturally occurring molecules with antimicrobial activity.
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molecular characterization of alpha lactalbumin folding variants that induce apoptosis in tumor cells
Journal of Biological Chemistry, 1999Co-Authors: Malin Svensson, Ann-kristin Mossberg, Anders Håkansson, Catharina Svanborg, Hemant Sabharwal, Peter H Lipniunas, Hakon Leffler, Sara LinseAbstract:This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk Alpha-Lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of Alpha-Lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule Alpha-Lactalbumin showed that the active fraction contains oligomers of Alpha-Lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve Alpha-Lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of Alpha-Lactalbumin.
Kunihiro Kuwajima - One of the best experts on this subject based on the ideXlab platform.
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structural characterization of the molten globule of alpha lactalbumin by solution x ray scattering
Protein Science, 1997Co-Authors: Mikio Kataoka, Kunihiro Kuwajima, Fumio Tokunaga, Yuji GotoAbstract:A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the Alpha-Lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of Alpha-Lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of Alpha-Lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the Alpha-Lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the Alpha-Lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.
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the molten globule state of alpha lactalbumin
The FASEB Journal, 1996Co-Authors: Kunihiro KuwajimaAbstract:The molten globule state of Alpha-Lactalbumin is the best-characterized folding intermediate of globular proteins and has been studied intensively by various spectroscopic and physiochemical techniques, including stopped-flow CD and fluorescence spectroscopies, a hydrogen-exchange technique, 1H-NMR spectroscopy, disulfide-exchange chemistry, site-directed mutagenesis, and calorimetric techniques. This review summarizes recent studies. Major findings about the structure of the molten globule state are: 1) It is highly heterogeneous, having a highly structured alpha-helical domain with the beta-sheet domain being significantly unfolded; and 2) it is not a nonspecific, collapsed polypeptide but already has a native-like tertiary fold. These structural characteristics are essential to fully understand the thermodynamic properties of the molten globule state which are described in connection with a recently proposed computational approach to predict the structure of the molten globule state of a protein. Mutant proteins in which the stability of the molten globule state was changed were constructed. Studies of the equilibrium unfolding and kinetic refolding of the mutant proteins will provide further insight into the molten globule state as a folding intermediate. In spite of an initial expectation that the structure recognized by an Escherichia coli chaperone, GroEL, is the molten globule, the interaction of GroEL with Alpha-Lactalbumin in the molten globule state is much weaker than the interaction with more unfolded states of Alpha-Lactalbumin, a disulfide-reduced form, and disulfide rearranged species.
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the molten globule state of alpha lactalbumin
The FASEB Journal, 1996Co-Authors: Kunihiro KuwajimaAbstract:The molten globule state of Alpha-Lactalbumin is the best-characterized folding intermediate of globular proteins and has been studied intensively by various spectroscopic and physiochemical techniques, including stopped-flow CD and fluorescence spectroscopies, a hydrogen-exchange technique, 1H-NMR spectroscopy, disulfide-exchange chemistry, site-directed mutagenesis, and calorimetric techniques. This review summarizes recent studies. Major findings about the structure of the molten globule state are: 1) It is highly heterogeneous, having a highly structured alpha-helical domain with the beta-sheet domain being significantly unfolded; and 2) it is not a nonspecific, collapsed polypeptide but already has a native-like tertiary fold. These structural characteristics are essential to fully understand the thermodynamic properties of the molten globule state which are described in connection with a recently proposed computational approach to predict the structure of the molten globule state of a protein. Mutan...