Final Report Summary - RENZI_FP7_IOF2007 (CryoEm structure of gamma secretase: a key component in Alzheimer neurodegenerative disease)
Project context and objectives
Gamma-secretase (g-sec) is a membrane protein complex. It is a 180 kDa tetrameric protease, assembled from a stable inactive trimeric core, composed of the catalytic sub-unit presenilin, the substrate binding protein nicastrin and the accessory protein APH1; the incorporation of the fourth component PEN2 (presenilin enhancer 2) triggers the endoproteolysis of presenilin resulting in an active tetrameric g-sec. By cleaving into the transmembrane domain of membrane-signalling precursors, g-sec is responsible for their proteolytic maturation; among these precursors the amyloid protein precursor (APP) is particularly important for its biomedical implications: its cleavage by g-sec results, in fact, in the production of the amyloid beta-peptides, which are implicated in Alzheimer's disease (AD).
The scientific aim of this project was to carry out structural studies of g-sec and to provide a 3-D reconstruction by single particle electron microscopy (EM). This is relevant, both in basic research to understand the as yet uncharacterised mechanism of intra-membrane proteolysis and in applied biomedical research for targeted drug design to interfere with the malfunctioning of g-sec in AD.
Work performed
The long-term goals were to enlarge the background of Dr Renzi's structural studies: previous X-ray crystallography studies, suitable to determine the structure of small proteins at high-resolution, would be implemented by the EM techniques, suitable to study large complexes at low to medium resolution. The combination of these two techniques would allow the study of large assemblies at high resolution. In addition a permanent collaboration between the European Union (EU) and the United States of America (USA) would be established.
Project results
Dr Renzi learnt to prepare specimens suitable for EM, to handle electron microscopes and to collect EM images; she learnt to reduce and process raw data, to carry out a 3-D reconstruction, both with and without an initial model, using the EM software (EMAN, iMagic, SPIDER, FReAlign, XMIPPS, eTomo, Chimera, etc.). Dr Renzi established a stable collaboration with the USAs institution by applying for grants in common projects.
Dr Renzi determined the 3-D reconstruction of a mature and catalytically active g-sec using single-particle cryo-EM. After assessing the quality of the sample by site-specific gold labelling, she collected EM images, and reduced and analysed raw data to carry out a 3-D reconstruction. G-sec has a cup-like shape with a lateral belt of about 45 A in height that encloses a water-accessible internal chamber. Active site labelling with a gold-coupled transition state analogue inhibitor suggested that the active site is inside this chamber. The structure thus suggests that the water-dependent proteolysis can have a place in the hydrophobic membrane, if intra-membrane proteases isolate their active site in a hydrophilic environment in their matrix while inserted in the membrane, to allocate the substrate.
Dr Renzi also collected EM images of the catalytically inactive trimeric core and carried out a 3-D reconstruction: the aim was to compare the tetramer and the trimer and to understand how the addition of PEN2 would enhance the activity of the complex. A comparison suggests that the incorporation of PEN-2 might contribute to the maturation of the active site architecture in enlarging the internal cavity and the accessibility of the active site to the substrate. The location of the surface cavities, their position relative to the internal chamber and, possibly, to the membrane, suggest as well a possible pathway for the substrate to access the active site and the cleavage products to be released. Project context and objectives
gamma-secretase (g-sec) is a membrane protein complex. It is a 180 kDa tetrameric protease, assembled from a stable inactive trimeric core, composed of the catalytic sub-unit presenilin, the substrate binding protein nicastrin and the accessory protein APH1; the incorporation of the fourth component PEN2 (presenilin enhancer 2) triggers the endoproteolysis of presenilin resulting in an active tetrameric g-sec. By cleaving into the transmembrane domain of membrane-signalling precursors, g-sec is responsible for their proteolytic maturation; among these precursors the amyloid protein precursor (APP) is particularly important for its biomedical implications: its cleavage by g-sec results, in fact, in the production of the amyloid beta-peptides, which are implicated in Alzheimer's disease (AD).
the scientific aim of this project was to carry out structural studies of g-sec and to provide a 3-D reconstruction by single particle electron microscopy (EM). This is relevant, both in basic research to understand the as yet uncharacterised mechanism of intra-membrane proteolysis and in applied biomedical research for targeted drug design to interfere with the malfunctioning of g-sec in AD.
work performed
the long-term goals were to enlarge the background of Dr Renzi's structural studies: previous X-ray crystallography studies, suitable to determine the structure of small proteins at high-resolution, would be implemented by the EM techniques, suitable to study large complexes at low to medium resolution. The combination of these two techniques would allow the study of large assemblies at high resolution. In addition a permanent collaboration between the European Union (EU) and the United States of America (USA) would be established.
project results
Dr Renzi learnt to prepare specimens suitable for EM, to handle electron microscopes and to collect EM images; she learnt to reduce and process raw data, to carry out a 3-D reconstruction, both with and without an initial model, using the EM software (EMAN, iMagic, SPIDER, FReAlign, XMIPPS, eTomo, Chimera, etc.). Dr Renzi established a stable collaboration with the USAs institution by applying for grants in common projects.
Dr Renzi determined the 3-D reconstruction of a mature and catalytically active g-sec using single-particle cryo-EM. After assessing the quality of the sample by site-specific gold labelling, she collected EM images, and reduced and analysed raw data to carry out a 3-D reconstruction. G-sec has a cup-like shape with a lateral belt of about 45 A in height that encloses a water-accessible internal chamber. Active site labelling with a gold-coupled transition state analogue inhibitor suggested that the active site is inside this chamber. The structure thus suggests that the water-dependent proteolysis can have a place in the hydrophobic membrane, if intra-membrane proteases isolate their active site in a hydrophilic environment in their matrix while inserted in the membrane, to allocate the substrate.
Dr Renzi also collected EM images of the catalytically inactive trimeric core and carried out a 3-D reconstruction: the aim was to compare the tetramer and the trimer and to understand how the addition of PEN2 would enhance the activity of the complex. A comparison suggests that the incorporation of PEN-2 might contribute to the maturation of the active site architecture in enlarging the internal cavity and the accessibility of the active site to the substrate. The location of the surface cavities, their position relative to the internal chamber and, possibly, to the membrane, suggest as well a possible pathway for the substrate to access the active site and the cleavage products to be released. Project context and objectives
gamma-secretase (g-sec) is a membrane protein complex. It is a 180 kDa tetrameric protease, assembled from a stable inactive trimeric core, composed of the catalytic sub-unit presenilin, the substrate binding protein nicastrin and the accessory protein APH1; the incorporation of the fourth component PEN2 (presenilin enhancer 2) triggers the endoproteolysis of presenilin resulting in an active tetrameric g-sec. By cleaving into the transmembrane domain of membrane-signalling precursors, g-sec is responsible for their proteolytic maturation; among these precursors the amyloid protein precursor (APP) is particularly important for its biomedical implications: its cleavage by g-sec results, in fact, in the production of the amyloid beta-peptides, which are implicated in Alzheimer's disease (AD).
the scientific aim of this project was to carry out structural studies of g-sec and to provide a 3-D reconstruction by single particle electron microscopy (EM). This is relevant, both in basic research to understand the as yet uncharacterised mechanism of intra-membrane proteolysis and in applied biomedical research for targeted drug design to interfere with the malfunctioning of g-sec in AD.
work performed
the long-term goals were to enlarge the background of Dr Renzi's structural studies: previous X-ray crystallography studies, suitable to determine the structure of small proteins at high-resolution, would be implemented by the EM techniques, suitable to study large complexes at low to medium resolution. The combination of these two techniques would allow the study of large assemblies at high resolution. In addition a permanent collaboration between the European Union (EU) and the United States of America (USA) would be established.
project results
Dr Renzi learnt to prepare specimens suitable for EM, to handle electron microscopes and to collect EM images; she learnt to reduce and process raw data, to carry out a 3-D reconstruction, both with and without an initial model, using the EM software (EMAN, iMagic, SPIDER, FReAlign, XMIPPS, eTomo, Chimera, etc.). Dr Renzi established a stable collaboration with the USAs institution by applying for grants in common projects.
Dr Renzi determined the 3-D reconstruction of a mature and catalytically active g-sec using single-particle cryo-EM. After assessing the quality of the sample by site-specific gold labelling, she collected EM images, and reduced and analysed raw data to carry out a 3-D reconstruction. G-sec has a cup-like shape with a lateral belt of about 45 A in height that encloses a water-accessible internal chamber. Active site labelling with a gold-coupled transition state analogue inhibitor suggested that the active site is inside this chamber. The structure thus suggests that the water-dependent proteolysis can have a place in the hydrophobic membrane, if intra-membrane proteases isolate their active site in a hydrophilic environment in their matrix while inserted in the membrane, to allocate the substrate.
Dr Renzi also collected EM images of the catalytically inactive trimeric core and carried out a 3-D reconstruction: the aim was to compare the tetramer and the trimer and to understand how the addition of PEN2 would enhance the activity of the complex. A comparison suggests that the incorporation of PEN-2 might contribute to the maturation of the active site architecture in enlarging the internal cavity and the accessibility of the active site to the substrate. The location of the surface cavities, their position relative to the internal chamber and, possibly, to the membrane, suggest as well a possible pathway for the substrate to access the active site and the cleavage products to be released.
Gamma-secretase (g-sec) is a membrane protein complex. It is a 180 kDa tetrameric protease, assembled from a stable inactive trimeric core, composed of the catalytic sub-unit presenilin, the substrate binding protein nicastrin and the accessory protein APH1; the incorporation of the fourth component PEN2 (presenilin enhancer 2) triggers the endoproteolysis of presenilin resulting in an active tetrameric g-sec. By cleaving into the transmembrane domain of membrane-signalling precursors, g-sec is responsible for their proteolytic maturation; among these precursors the amyloid protein precursor (APP) is particularly important for its biomedical implications: its cleavage by g-sec results, in fact, in the production of the amyloid beta-peptides, which are implicated in Alzheimer's disease (AD).
The scientific aim of this project was to carry out structural studies of g-sec and to provide a 3-D reconstruction by single particle electron microscopy (EM). This is relevant, both in basic research to understand the as yet uncharacterised mechanism of intra-membrane proteolysis and in applied biomedical research for targeted drug design to interfere with the malfunctioning of g-sec in AD.
Work performed
The long-term goals were to enlarge the background of Dr Renzi's structural studies: previous X-ray crystallography studies, suitable to determine the structure of small proteins at high-resolution, would be implemented by the EM techniques, suitable to study large complexes at low to medium resolution. The combination of these two techniques would allow the study of large assemblies at high resolution. In addition a permanent collaboration between the European Union (EU) and the United States of America (USA) would be established.
Project results
Dr Renzi learnt to prepare specimens suitable for EM, to handle electron microscopes and to collect EM images; she learnt to reduce and process raw data, to carry out a 3-D reconstruction, both with and without an initial model, using the EM software (EMAN, iMagic, SPIDER, FReAlign, XMIPPS, eTomo, Chimera, etc.). Dr Renzi established a stable collaboration with the USAs institution by applying for grants in common projects.
Dr Renzi determined the 3-D reconstruction of a mature and catalytically active g-sec using single-particle cryo-EM. After assessing the quality of the sample by site-specific gold labelling, she collected EM images, and reduced and analysed raw data to carry out a 3-D reconstruction. G-sec has a cup-like shape with a lateral belt of about 45 A in height that encloses a water-accessible internal chamber. Active site labelling with a gold-coupled transition state analogue inhibitor suggested that the active site is inside this chamber. The structure thus suggests that the water-dependent proteolysis can have a place in the hydrophobic membrane, if intra-membrane proteases isolate their active site in a hydrophilic environment in their matrix while inserted in the membrane, to allocate the substrate.
Dr Renzi also collected EM images of the catalytically inactive trimeric core and carried out a 3-D reconstruction: the aim was to compare the tetramer and the trimer and to understand how the addition of PEN2 would enhance the activity of the complex. A comparison suggests that the incorporation of PEN-2 might contribute to the maturation of the active site architecture in enlarging the internal cavity and the accessibility of the active site to the substrate. The location of the surface cavities, their position relative to the internal chamber and, possibly, to the membrane, suggest as well a possible pathway for the substrate to access the active site and the cleavage products to be released. Project context and objectives
gamma-secretase (g-sec) is a membrane protein complex. It is a 180 kDa tetrameric protease, assembled from a stable inactive trimeric core, composed of the catalytic sub-unit presenilin, the substrate binding protein nicastrin and the accessory protein APH1; the incorporation of the fourth component PEN2 (presenilin enhancer 2) triggers the endoproteolysis of presenilin resulting in an active tetrameric g-sec. By cleaving into the transmembrane domain of membrane-signalling precursors, g-sec is responsible for their proteolytic maturation; among these precursors the amyloid protein precursor (APP) is particularly important for its biomedical implications: its cleavage by g-sec results, in fact, in the production of the amyloid beta-peptides, which are implicated in Alzheimer's disease (AD).
the scientific aim of this project was to carry out structural studies of g-sec and to provide a 3-D reconstruction by single particle electron microscopy (EM). This is relevant, both in basic research to understand the as yet uncharacterised mechanism of intra-membrane proteolysis and in applied biomedical research for targeted drug design to interfere with the malfunctioning of g-sec in AD.
work performed
the long-term goals were to enlarge the background of Dr Renzi's structural studies: previous X-ray crystallography studies, suitable to determine the structure of small proteins at high-resolution, would be implemented by the EM techniques, suitable to study large complexes at low to medium resolution. The combination of these two techniques would allow the study of large assemblies at high resolution. In addition a permanent collaboration between the European Union (EU) and the United States of America (USA) would be established.
project results
Dr Renzi learnt to prepare specimens suitable for EM, to handle electron microscopes and to collect EM images; she learnt to reduce and process raw data, to carry out a 3-D reconstruction, both with and without an initial model, using the EM software (EMAN, iMagic, SPIDER, FReAlign, XMIPPS, eTomo, Chimera, etc.). Dr Renzi established a stable collaboration with the USAs institution by applying for grants in common projects.
Dr Renzi determined the 3-D reconstruction of a mature and catalytically active g-sec using single-particle cryo-EM. After assessing the quality of the sample by site-specific gold labelling, she collected EM images, and reduced and analysed raw data to carry out a 3-D reconstruction. G-sec has a cup-like shape with a lateral belt of about 45 A in height that encloses a water-accessible internal chamber. Active site labelling with a gold-coupled transition state analogue inhibitor suggested that the active site is inside this chamber. The structure thus suggests that the water-dependent proteolysis can have a place in the hydrophobic membrane, if intra-membrane proteases isolate their active site in a hydrophilic environment in their matrix while inserted in the membrane, to allocate the substrate.
Dr Renzi also collected EM images of the catalytically inactive trimeric core and carried out a 3-D reconstruction: the aim was to compare the tetramer and the trimer and to understand how the addition of PEN2 would enhance the activity of the complex. A comparison suggests that the incorporation of PEN-2 might contribute to the maturation of the active site architecture in enlarging the internal cavity and the accessibility of the active site to the substrate. The location of the surface cavities, their position relative to the internal chamber and, possibly, to the membrane, suggest as well a possible pathway for the substrate to access the active site and the cleavage products to be released. Project context and objectives
gamma-secretase (g-sec) is a membrane protein complex. It is a 180 kDa tetrameric protease, assembled from a stable inactive trimeric core, composed of the catalytic sub-unit presenilin, the substrate binding protein nicastrin and the accessory protein APH1; the incorporation of the fourth component PEN2 (presenilin enhancer 2) triggers the endoproteolysis of presenilin resulting in an active tetrameric g-sec. By cleaving into the transmembrane domain of membrane-signalling precursors, g-sec is responsible for their proteolytic maturation; among these precursors the amyloid protein precursor (APP) is particularly important for its biomedical implications: its cleavage by g-sec results, in fact, in the production of the amyloid beta-peptides, which are implicated in Alzheimer's disease (AD).
the scientific aim of this project was to carry out structural studies of g-sec and to provide a 3-D reconstruction by single particle electron microscopy (EM). This is relevant, both in basic research to understand the as yet uncharacterised mechanism of intra-membrane proteolysis and in applied biomedical research for targeted drug design to interfere with the malfunctioning of g-sec in AD.
work performed
the long-term goals were to enlarge the background of Dr Renzi's structural studies: previous X-ray crystallography studies, suitable to determine the structure of small proteins at high-resolution, would be implemented by the EM techniques, suitable to study large complexes at low to medium resolution. The combination of these two techniques would allow the study of large assemblies at high resolution. In addition a permanent collaboration between the European Union (EU) and the United States of America (USA) would be established.
project results
Dr Renzi learnt to prepare specimens suitable for EM, to handle electron microscopes and to collect EM images; she learnt to reduce and process raw data, to carry out a 3-D reconstruction, both with and without an initial model, using the EM software (EMAN, iMagic, SPIDER, FReAlign, XMIPPS, eTomo, Chimera, etc.). Dr Renzi established a stable collaboration with the USAs institution by applying for grants in common projects.
Dr Renzi determined the 3-D reconstruction of a mature and catalytically active g-sec using single-particle cryo-EM. After assessing the quality of the sample by site-specific gold labelling, she collected EM images, and reduced and analysed raw data to carry out a 3-D reconstruction. G-sec has a cup-like shape with a lateral belt of about 45 A in height that encloses a water-accessible internal chamber. Active site labelling with a gold-coupled transition state analogue inhibitor suggested that the active site is inside this chamber. The structure thus suggests that the water-dependent proteolysis can have a place in the hydrophobic membrane, if intra-membrane proteases isolate their active site in a hydrophilic environment in their matrix while inserted in the membrane, to allocate the substrate.
Dr Renzi also collected EM images of the catalytically inactive trimeric core and carried out a 3-D reconstruction: the aim was to compare the tetramer and the trimer and to understand how the addition of PEN2 would enhance the activity of the complex. A comparison suggests that the incorporation of PEN-2 might contribute to the maturation of the active site architecture in enlarging the internal cavity and the accessibility of the active site to the substrate. The location of the surface cavities, their position relative to the internal chamber and, possibly, to the membrane, suggest as well a possible pathway for the substrate to access the active site and the cleavage products to be released.