The Experts below are selected from a list of 2949 Experts worldwide ranked by ideXlab platform
Pascal Leterme - One of the best experts on this subject based on the ideXlab platform.
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British Journal of Nutrition (2009), 101, 1324–1332 q The Authors 2008
2014Co-Authors: Carlos A Montoya, Stephen Beebe, Jean Paul Lalles, Wolfgang B. Souffrant, Daniel Molle, Pascal LetermeAbstract:doi:10.1017/S0007114508060790 Susceptibility of Phaseolin (Phaseolus vulgaris) subunits to trypsinolysis and influence of dietary level of raw Phaseolin on protein digestion in the small intestine of rat
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Phaseolin from phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats
British Journal of Nutrition, 2010Co-Authors: Carlos A Montoya, Pascal Leterme, Stephen Beebe, Veronique Rome, Jean Claustre, Jean Paul LallesAbstract:Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated Phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated Phaseolin influences mucin flow and gene expression, and that Phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67 % of unheated and 67 % of heated Phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated Phaseolin (P < 0·05). Unheated Phaseolin increased N flow in ileum, colon and faeces (P < 0·05), and reduced apparent N digestibility linearly (P < 0·01). Heat treatment reversed all these changes (P < 0·05 to < 0·001), except mucin flow. The expressions of Muc mRNA in gut tissues were influenced by dietary Phaseolin level (ileum and colon: Muc3 and Muc4) and thermal treatment (ileum: Muc2; colon: Muc2, Muc3, Muc4 and Tff3) (P < 0·05 to 0·001). In conclusion, Phaseolin modulates mucin flow and Muc gene expression along the intestines differentially.
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Phaseolin diversity as a possible strategy to improve the nutritional value of common beans phaseolus vulgaris
Food Research International, 2010Co-Authors: Carlos A Montoya, Jean Paul Lalles, Stephen Beebe, Pascal LetermeAbstract:Abstract This article proposes a new way to improve the protein quality of the common bean (Phaseolus vulgaris). It is based on the natural variability found in the different types of Phaseolin, its main storage protein (40–50% of the total protein). Despite the fact that it is deficient in methionine content, Phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2–6) and molecular weight (40–54 kDa) have been analyzed. The similarity of the amino acid composition among Phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in Phaseolin susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based on highly-digestible Phaseolin types could lead to the production of beans with higher protein quality.
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Phaseolin diversity as a possible strategy to improve the nutritional value of common beans (Phaseolus vulgaris): a review
Food Research International, 2010Co-Authors: Carlos Montoya, Jean Paul Lalles, Stephen Beebe, Pascal LetermeAbstract:This article proposes a new way to improve the protein quality Of the common bean (Phaseolus vulgaris). it is based on the natural variability found in the different types of Phaseolin, its main storage protein (40-50% of the total protein). Despite the fact that it is deficient in methionine content, Phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2-6) and molecular weight (40-54 kDa) have been analyzed. The similarity of the amino acid composition among Phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in Phaseolin Susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based oil highly-digestible Phaseolin types could lead to the production of beans with higher protein quality. (C) 2009 Published by Elsevier Ltd.
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Phaseolin from Phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats
British Journal of Nutrition, 2010Co-Authors: Carlos Alexander Montoya, Pascal Leterme, Stephen Beebe, Veronique Rome, Jean Claustre, Jean Paul LallesAbstract:Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated Phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated Phaseolin influences mucin flow and gene expression, and that Phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67% of unheated and 67% of heated Phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated Phaseolin (P
Jean Paul Lalles - One of the best experts on this subject based on the ideXlab platform.
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British Journal of Nutrition (2009), 101, 1324–1332 q The Authors 2008
2014Co-Authors: Carlos A Montoya, Stephen Beebe, Jean Paul Lalles, Wolfgang B. Souffrant, Daniel Molle, Pascal LetermeAbstract:doi:10.1017/S0007114508060790 Susceptibility of Phaseolin (Phaseolus vulgaris) subunits to trypsinolysis and influence of dietary level of raw Phaseolin on protein digestion in the small intestine of rat
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Phaseolin from phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats
British Journal of Nutrition, 2010Co-Authors: Carlos A Montoya, Pascal Leterme, Stephen Beebe, Veronique Rome, Jean Claustre, Jean Paul LallesAbstract:Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated Phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated Phaseolin influences mucin flow and gene expression, and that Phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67 % of unheated and 67 % of heated Phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated Phaseolin (P < 0·05). Unheated Phaseolin increased N flow in ileum, colon and faeces (P < 0·05), and reduced apparent N digestibility linearly (P < 0·01). Heat treatment reversed all these changes (P < 0·05 to < 0·001), except mucin flow. The expressions of Muc mRNA in gut tissues were influenced by dietary Phaseolin level (ileum and colon: Muc3 and Muc4) and thermal treatment (ileum: Muc2; colon: Muc2, Muc3, Muc4 and Tff3) (P < 0·05 to 0·001). In conclusion, Phaseolin modulates mucin flow and Muc gene expression along the intestines differentially.
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Phaseolin diversity as a possible strategy to improve the nutritional value of common beans phaseolus vulgaris
Food Research International, 2010Co-Authors: Carlos A Montoya, Jean Paul Lalles, Stephen Beebe, Pascal LetermeAbstract:Abstract This article proposes a new way to improve the protein quality of the common bean (Phaseolus vulgaris). It is based on the natural variability found in the different types of Phaseolin, its main storage protein (40–50% of the total protein). Despite the fact that it is deficient in methionine content, Phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2–6) and molecular weight (40–54 kDa) have been analyzed. The similarity of the amino acid composition among Phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in Phaseolin susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based on highly-digestible Phaseolin types could lead to the production of beans with higher protein quality.
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Phaseolin diversity as a possible strategy to improve the nutritional value of common beans (Phaseolus vulgaris): a review
Food Research International, 2010Co-Authors: Carlos Montoya, Jean Paul Lalles, Stephen Beebe, Pascal LetermeAbstract:This article proposes a new way to improve the protein quality Of the common bean (Phaseolus vulgaris). it is based on the natural variability found in the different types of Phaseolin, its main storage protein (40-50% of the total protein). Despite the fact that it is deficient in methionine content, Phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2-6) and molecular weight (40-54 kDa) have been analyzed. The similarity of the amino acid composition among Phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in Phaseolin Susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based oil highly-digestible Phaseolin types could lead to the production of beans with higher protein quality. (C) 2009 Published by Elsevier Ltd.
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Phaseolin from Phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats
British Journal of Nutrition, 2010Co-Authors: Carlos Alexander Montoya, Pascal Leterme, Stephen Beebe, Veronique Rome, Jean Claustre, Jean Paul LallesAbstract:Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated Phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated Phaseolin influences mucin flow and gene expression, and that Phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67% of unheated and 67% of heated Phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated Phaseolin (P
Carlos A Montoya - One of the best experts on this subject based on the ideXlab platform.
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British Journal of Nutrition (2009), 101, 1324–1332 q The Authors 2008
2014Co-Authors: Carlos A Montoya, Stephen Beebe, Jean Paul Lalles, Wolfgang B. Souffrant, Daniel Molle, Pascal LetermeAbstract:doi:10.1017/S0007114508060790 Susceptibility of Phaseolin (Phaseolus vulgaris) subunits to trypsinolysis and influence of dietary level of raw Phaseolin on protein digestion in the small intestine of rat
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Phaseolin from phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats
British Journal of Nutrition, 2010Co-Authors: Carlos A Montoya, Pascal Leterme, Stephen Beebe, Veronique Rome, Jean Claustre, Jean Paul LallesAbstract:Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated Phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated Phaseolin influences mucin flow and gene expression, and that Phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67 % of unheated and 67 % of heated Phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated Phaseolin (P < 0·05). Unheated Phaseolin increased N flow in ileum, colon and faeces (P < 0·05), and reduced apparent N digestibility linearly (P < 0·01). Heat treatment reversed all these changes (P < 0·05 to < 0·001), except mucin flow. The expressions of Muc mRNA in gut tissues were influenced by dietary Phaseolin level (ileum and colon: Muc3 and Muc4) and thermal treatment (ileum: Muc2; colon: Muc2, Muc3, Muc4 and Tff3) (P < 0·05 to 0·001). In conclusion, Phaseolin modulates mucin flow and Muc gene expression along the intestines differentially.
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Phaseolin diversity as a possible strategy to improve the nutritional value of common beans phaseolus vulgaris
Food Research International, 2010Co-Authors: Carlos A Montoya, Jean Paul Lalles, Stephen Beebe, Pascal LetermeAbstract:Abstract This article proposes a new way to improve the protein quality of the common bean (Phaseolus vulgaris). It is based on the natural variability found in the different types of Phaseolin, its main storage protein (40–50% of the total protein). Despite the fact that it is deficient in methionine content, Phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2–6) and molecular weight (40–54 kDa) have been analyzed. The similarity of the amino acid composition among Phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in Phaseolin susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based on highly-digestible Phaseolin types could lead to the production of beans with higher protein quality.
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susceptibility of Phaseolin phaseolus vulgaris subunits to trypsinolysis and influence of dietary level of raw Phaseolin on protein digestion in the small intestine of rats
British Journal of Nutrition, 2009Co-Authors: Carlos A Montoya, Stephen Beebe, Jean Paul Lalles, Wolfgang B. Souffrant, Daniel Molle, Pascal LetermeAbstract:The aim of the present work was (a) to investigate trypsinolysis of denatured purified T Phaseolin (Phaseolus vulgaris) subunits by MS and (b) to test the effect of raw T Phaseolin inclusion level in diets fed chronically to rats on digestion in the small intestine. The diets contained casein as the sole protein source, or casein substituted with 33, 67 and 100 % of purified T Phaseolin. Rats were fed for 10 d and then euthanised. Digesta and tissues from the first and second halves of the small intestine were prepared for electrophoresis, immunoblotting and densitometry. alpha-Phaseolin subunit for the T Phaseolin was more resistant to trypsinolysis than beta-Phaseolin subunit. Nearly intact Phaseolin subunits (molecular weight, MW 44-54 kDa) and partially digested Phaseolin fragments (MW 17-19 and 20-24 kDa) were identified in small intestinal digesta. The concentration of intact Phaseolin and of most undigested Phaseolin fragments in digesta increased in the second half of the small intestine with increasing Phaseolin intake (P < 0.05-0.01). The concentration of Phaseolin fragments of a MW of 21-22.5 and 23-24.5 kDa in the mucosa increased linearly (P = 0.016-0.084) when the level of the T Phaseolin was increased in the diet. In conclusion, the present work provides evidence that denatured T Phaseolin subunits display different trypsinolysis patterns in vitro. Moreover, a high intake of raw T Phaseolin impacts digestion in the small intestine of rats.
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Susceptibility of Phaseolin (Phaseolus vulgaris) subunits to trypsinolysis and influence of dietary level of raw Phaseolin on protein digestion in the small intestine of rats
British Journal of Nutrition, 2009Co-Authors: Carlos A Montoya, Stephen Beebe, Jean Paul Lalles, Wolfgang B. Souffrant, Daniel Molle, Pascal LetermeAbstract:The aim of the present work was (a) to investigate trypsinolysis of denatured purified T Phaseolin (Phaseolus vulgaris) subunits by MS and (b) to test the effect of raw T Phaseolin inclusion level in diets fed chronically to rats on digestion in the small intestine. The diets contained casein as the sole protein source, or casein substituted with 33, 67 and 100% of purified T Phaseolin. Rats were fed for 10 d and then euthanised. Digesta and tissues from the first and second halves of the small intestine were prepared for electrophoresis, immunoblotting and densitometry. a-Phaseolin subunit for the T Phaseolin was more resistant to trypsinolysis than b-Phaseolin subunit. Nearly intact Phaseolin subunits (molecular weight, MW 44-54 kDa) and partially digested Phaseolin fragments (MW 17-19 and 20-24 kDa) were identified in small intestinal digesta. The concentration of intact Phaseolin and of most undigested Phaseolin fragments in digesta increased in the second half of the small intestine with increasing Phaseolin intake (P,0*05-0*01). The concentration of Phaseolin fragments of a MW of 21-22*5 and 23-24*5 kDa in the mucosa increased linearly (P¼0*016-0*084) when the level of the T Phaseolin was increased in the diet. In conclusion, the present work provides evidence that denatured T Phaseolin subunits display different trypsinolysis patterns in vitro. Moreover, a high intake of raw T Phaseolin impacts digestion in the small intestine of rats.
Stephen Beebe - One of the best experts on this subject based on the ideXlab platform.
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British Journal of Nutrition (2009), 101, 1324–1332 q The Authors 2008
2014Co-Authors: Carlos A Montoya, Stephen Beebe, Jean Paul Lalles, Wolfgang B. Souffrant, Daniel Molle, Pascal LetermeAbstract:doi:10.1017/S0007114508060790 Susceptibility of Phaseolin (Phaseolus vulgaris) subunits to trypsinolysis and influence of dietary level of raw Phaseolin on protein digestion in the small intestine of rat
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Phaseolin from phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats
British Journal of Nutrition, 2010Co-Authors: Carlos A Montoya, Pascal Leterme, Stephen Beebe, Veronique Rome, Jean Claustre, Jean Paul LallesAbstract:Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated Phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated Phaseolin influences mucin flow and gene expression, and that Phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67 % of unheated and 67 % of heated Phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated Phaseolin (P < 0·05). Unheated Phaseolin increased N flow in ileum, colon and faeces (P < 0·05), and reduced apparent N digestibility linearly (P < 0·01). Heat treatment reversed all these changes (P < 0·05 to < 0·001), except mucin flow. The expressions of Muc mRNA in gut tissues were influenced by dietary Phaseolin level (ileum and colon: Muc3 and Muc4) and thermal treatment (ileum: Muc2; colon: Muc2, Muc3, Muc4 and Tff3) (P < 0·05 to 0·001). In conclusion, Phaseolin modulates mucin flow and Muc gene expression along the intestines differentially.
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Phaseolin diversity as a possible strategy to improve the nutritional value of common beans phaseolus vulgaris
Food Research International, 2010Co-Authors: Carlos A Montoya, Jean Paul Lalles, Stephen Beebe, Pascal LetermeAbstract:Abstract This article proposes a new way to improve the protein quality of the common bean (Phaseolus vulgaris). It is based on the natural variability found in the different types of Phaseolin, its main storage protein (40–50% of the total protein). Despite the fact that it is deficient in methionine content, Phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2–6) and molecular weight (40–54 kDa) have been analyzed. The similarity of the amino acid composition among Phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in Phaseolin susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based on highly-digestible Phaseolin types could lead to the production of beans with higher protein quality.
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Phaseolin diversity as a possible strategy to improve the nutritional value of common beans (Phaseolus vulgaris): a review
Food Research International, 2010Co-Authors: Carlos Montoya, Jean Paul Lalles, Stephen Beebe, Pascal LetermeAbstract:This article proposes a new way to improve the protein quality Of the common bean (Phaseolus vulgaris). it is based on the natural variability found in the different types of Phaseolin, its main storage protein (40-50% of the total protein). Despite the fact that it is deficient in methionine content, Phaseolin still represents the main source of that amino acid in the seed. More than 40 genetic variants, differing in subunit number (2-6) and molecular weight (40-54 kDa) have been analyzed. The similarity of the amino acid composition among Phaseolins, suggests that a nutritional improvement cannot be expected from that side. Conversely, important variation in Phaseolin Susceptibility to proteolysis (ranging from 57% to 96% after cooking) has been observed, increasing the theoretical availability of methionine by up to 37%. Therefore, breeding programs based oil highly-digestible Phaseolin types could lead to the production of beans with higher protein quality. (C) 2009 Published by Elsevier Ltd.
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Phaseolin from Phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats
British Journal of Nutrition, 2010Co-Authors: Carlos Alexander Montoya, Pascal Leterme, Stephen Beebe, Veronique Rome, Jean Claustre, Jean Paul LallesAbstract:Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated Phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated Phaseolin influences mucin flow and gene expression, and that Phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67% of unheated and 67% of heated Phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated Phaseolin (P
Alessandro Vitale - One of the best experts on this subject based on the ideXlab platform.
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an engineered c terminal disulfide bond can partially replace the Phaseolin vacuolar sorting signal
Plant Journal, 2010Co-Authors: Andrea Pompa, Alessandro Vitale, Francesca De Marchis, Sergio Arcioni, Michele BellucciAbstract:Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein Phaseolin and the N-terminal half of the Zea mays prolamin γ-zein forms interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type Phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the Phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated Phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Δ418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Δ418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of Phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.
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the Phaseolin vacuolar sorting signal promotes transient strong membrane association and aggregation of the bean storage protein in transgenic tobacco
Journal of Experimental Botany, 2005Co-Authors: Silvana Castelli, Alessandro VitaleAbstract:Vacuolar storage proteins of the 7S class are co-translationally introduced into the endoplasmic reticulum and reach storage vacuoles via the Golgi complex and dense vesicles. The signal for vacuolar sorting of one of these proteins, Phaseolin of Phaseolus vulgaris, consists of a four-amino acid hydrophobic propeptide at the C-terminus. When this sequence is deleted, Phaseolin is secreted instead of being sorted to vacuoles. It is shown here that in transgenic tobacco plants newly-synthesized Phaseolin has unusual affinity to membranes and forms SDS-resistant aggregates, but mutated Phaseolin polypeptides that are either secreted or defective in assembly do not have these characteristics. Association to membranes and aggregation are transient events: Phaseolin accumulated in vacuoles is soluble in the absence of detergents and is not aggregated. Association to membranes starts before the Phaseolin glycan acquires a complex structure and therefore before the protein reaches the medial or trans-cisternae of the Golgi complex. These results support the hypothesis of a relationship between aggregation and vacuolar sorting of Phaseolin and indicate that sorting may start in early compartments of the secretory pathway.
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a Phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone bip
The Plant Cell, 2003Co-Authors: Ombretta Foresti, Lorenzo Frigerio, Maddalena De Virgilio, Heidi Holkeri, Stefano Vavassori, Alessandro VitaleAbstract:The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein Phaseolin associate with BiP and that Phaseolin trimerization in the ER abolishes such interactions. Using different Phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two α-helical regions of polypeptide contact in Phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with Phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters Phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of Phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for Phaseolin assembly in vivo and contains one potential BiP binding site.
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influence of kdel on the fate of trimeric or assembly defective Phaseolin selective use of an alternative route to vacuoles
The Plant Cell, 2001Co-Authors: Lorenzo Frigerio, Alessandra Prada, Alessandra Pastres, Alessandro VitaleAbstract:The tetrapeptide KDEL is commonly found at the C terminus of soluble proteins of the endoplasmic reticulum (ER), and it contributes to their localization by interacting with a receptor that recycles between the Golgi complex and the ER. We investigated the effects of the addition of KDEL to Phaseolin, a protein normally delivered from the ER to storage vacuoles via the Golgi complex. We show that KDEL prevents acquisition of trans-Golgi–specific glycan modifications and causes interactions with the chaperone BiP that are distinct from the ones between BiP and defective proteins. KDEL markedly increases the stability of Phaseolin, but a small proportion of Phaseolin-KDEL slowly reaches the vacuole without undergoing Golgi-mediated glycan modifications, in a process that can be inhibited by brefeldin A but not monensin. Our results indicate that KDEL can operate with high efficiency before proteins can reach the late Golgi cisternae but allows or promotes delivery to vacuoles via an alternative mechanism. However, addition of KDEL does not alter the destiny of an assembly-defective form of Phaseolin, suggesting that the plant ER quality control mechanism is dominant over KDEL effects.
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sorting of Phaseolin to the vacuole is saturable and requires a short c terminal peptide
The Plant Cell, 1998Co-Authors: Lorenzo Frigerio, Maddalena De Virgilio, Alessandra Prada, Franco Faoro, Alessandro VitaleAbstract:Phaseolin, one of the major legume proteins for human nutrition, is a trimeric glycoprotein of the 7S class that accumulates in the protein storage vacuoles of common bean. Phaseolin is cotranslationally introduced into the lumen of the endoplasmic reticulum; from there, it is transported through the Golgi complex to the storage vacuoles. Phaseolin is also transported to the vacuole in vegetative tissues of transgenic plants. By transient and permanent expression in tobacco leaf cells, we show here that vacuolar sorting of Phaseolin is saturable and that saturation leads to Golgi-mediated secretion from the cell. A mutated Phaseolin, in which the four C-terminal residues (Ala, Phe, Val, and Tyr) were deleted, efficiently formed trimers but was secreted entirely outside of the cells in transgenic tobacco leaves, indicating that the deleted sequence contains information necessary for interactions with the saturable vacuolar sorting machinery. In the apoplast, the secreted Phaseolin remained intact; this is similar to what occurs to wild-type Phaseolin in bean storage vacuoles, whereas in vegetative vacuoles of transgenic plants, the storage protein is fragmented.