Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids
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Evaluation Summary:
This study sheds light on the formation of Prolactin/Galanin functional amyloids and their storage in secretory granules of the anterior pituitary gland. This study sharpens our understanding of the regulation of hormonal release from the pituitary gland. It will be of interest to the fields of endocrinology, neurobiology and cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Synergistic-aggregation and cross-seeding by two different proteins/peptides in the amyloid aggregation are well evident in various neurological disorders including Alzheimer’s disease. Here, we show co-storage of human Prolactin (PRL), which is associated with lactation in mammals, and neuropeptide galanin (GAL) as functional amyloids in secretory granules (SGs) of the female rat. Using a wide variety of biophysical studies, we show that irrespective of the difference in sequence and structure, both hormones facilitate their synergic aggregation to amyloid fibrils. Although each hormone possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation by PRL seeds and the inability of cross seeding by mixed fibrils suggest tight regulation of functional amyloid formation by these hormones for their efficient storage in SGs. Further, the faster release of functional hormones from mixed fibrils compared to the corresponding individual amyloid, suggests a novel mechanism of heterologous amyloid formation in functional amyloids of SGs in the pituitary.
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Author Response
Reviewer #1(Public Review)
Maji et al demonstrate co-storage of prolactin (PRL) and galanin (GAL) as functional amyloids in secretory granules of the female rat. In a series of detailed experiments, they show that both hormones promote their aggregation to amyloid. They show that PRL and GAL co-localize in the pituitary and that there is co-fibril formation, forming a new type of hybrid fibril. They further demonstrate that there is a unidirectional cross-seeding of GAL aggregation for PRL seeds, while cross seeding by mixed fibrils does not occur. Molecular dynamic studies show that co-aggregation of PRL and GAL induce the formation of a β-sheet at the protein surface. Overall, more efficient storage of the hormones in secretory granules is demonstrated, as well as faster release, as compared to the homotypic …
Author Response
Reviewer #1(Public Review)
Maji et al demonstrate co-storage of prolactin (PRL) and galanin (GAL) as functional amyloids in secretory granules of the female rat. In a series of detailed experiments, they show that both hormones promote their aggregation to amyloid. They show that PRL and GAL co-localize in the pituitary and that there is co-fibril formation, forming a new type of hybrid fibril. They further demonstrate that there is a unidirectional cross-seeding of GAL aggregation for PRL seeds, while cross seeding by mixed fibrils does not occur. Molecular dynamic studies show that co-aggregation of PRL and GAL induce the formation of a β-sheet at the protein surface. Overall, more efficient storage of the hormones in secretory granules is demonstrated, as well as faster release, as compared to the homotypic counterparts. Strengths include the rigorous techniques that were used, including biophysical techniques with transmission electron microscopy and the use of molecular dynamics to delineate the mechanism of PRL and GAL interactions at the atomic level. An additional strength is the novel observation of the unidirectional, heterotypic templating competency of PRL fibril seeds for GAL monomers, inducing GAL fibril formation.
We appreciate your comments and thank you for providing deep insight into our manuscript and stating the importance of our findings.
Reviewer #2 (Public Review):
Research on peptide hormones released from the Pituitary, including Prolactin, has shown that the hormones are stored as functional amyloids. Furthermore, it is well established that Prolactin and Galanin are co-stored in secretory granules of the anterior pituitary until they are released into the bloodstream. However, the mechanism by which hormones are stored and released remains a mystery. This study describes the co-aggregation and functional heterotypic amyloid formation of Prolactin and neuropeptide Galanin in secretory granules. This study suggests that the Prolactin and Galanin interact with each other at high specificity and form functional amyloids. These functional amyloids are heterotypic. Moreover, they demonstrated that Prolactin-Galanin amyloids can form surface-induced secondary fibrils on the surfaces of others. Galanin forms secondary fibrils on Prolactin seeds and Prolactin does not form secondary fibrils on Galanin seeds, indicating that this process occurs in a highly regulated manner. Additionally, they analyzed the release of hormone monomers from amyloids in vitro. They found that Prolactin-Galanin functional amyloids are released faster than amyloids formed by Prolactin or Galanin homotypic fibrils. To understand the interactions between Prolactin and Galanin at the atomic level, they have also performed molecular dynamics simulations and docking studies.
A high point of the study is the identification of Prolactin's capability to cross-seed Galanin. This causes amyloid fibrils to be formed. However, in contrast, the Galanin failed in cross-seed the Prolactin. It emphasizes the specificity and regulation in functional amyloid formation. Additionally, the understanding of the interactions between Prolactin and Galanin at the atomic level from MD simulations strengthens the findings. The results of this study did not confirm the possibility of heteromeric fibril formation.
In this study, the authors succeeded in achieving their goals and their conclusions were backed up by their results.
Undoubtedly, this work will have a significant impact on the field of endocrinology and protein aggregation. By studying secretory granules of the pituitary gland, researchers have successfully stepped one step closer to understanding peptide hormone synthesis and release.
We appreciate you for the elaborate analysis of our manuscript and for commenting on the relevance of our findings.
Reviewer #3 (Public Review):
Maji and coworkers present a tour de force study of the coaggregation of two hormones, prolactin and galanin. Protein aggregation in vivo is much more of a "messy" affair than in the tidy lab of an Eppendorf tube and the authors demonstrate an intimate collaboration between these two hormones in the aggregation process. Their work ranges from IHC of tissue slices over experimental biophysics to computational studies, presented in a clear, user-friendly and illustrative manner and overall, the conclusions are sound. I found the cartoon diagrams in various figures particularly helpful and of high quality.
Whether their conclusions can be extended to higher-order complexes between multiple hormones (even closer to real life) is the next question to address - but the mind boggles at the number of possibilities to explore.
We thank you for your detailed analysis of the studies in our manuscript and for highlighting the importance of our work.
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Evaluation Summary:
This study sheds light on the formation of Prolactin/Galanin functional amyloids and their storage in secretory granules of the anterior pituitary gland. This study sharpens our understanding of the regulation of hormonal release from the pituitary gland. It will be of interest to the fields of endocrinology, neurobiology and cancer.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Maji et al demonstrate co-storage of prolactin (PRL) and galanin (GAL) as functional amyloids in secretory granules of the female rat. In a series of detailed experiments, they show that both hormones promote their aggregation to amyloid. They show that PRL and GAL co-localize in the pituitary and that there is co-fibril formation, forming a new type of hybrid fibril. They further demonstrate that there is a unidirectional cross-seeding of GAL aggregation for PRL seeds, while cross seeding by mixed fibrils does not occur. Molecular dynamic studies show that co-aggregation of PRL and GAL induce the formation of a β-sheet at the protein surface. Overall, more efficient storage of the hormones in secretory granules is demonstrated, as well as faster release, as compared to the homotypic counterparts. Strengths …
Reviewer #1 (Public Review):
Maji et al demonstrate co-storage of prolactin (PRL) and galanin (GAL) as functional amyloids in secretory granules of the female rat. In a series of detailed experiments, they show that both hormones promote their aggregation to amyloid. They show that PRL and GAL co-localize in the pituitary and that there is co-fibril formation, forming a new type of hybrid fibril. They further demonstrate that there is a unidirectional cross-seeding of GAL aggregation for PRL seeds, while cross seeding by mixed fibrils does not occur. Molecular dynamic studies show that co-aggregation of PRL and GAL induce the formation of a β-sheet at the protein surface. Overall, more efficient storage of the hormones in secretory granules is demonstrated, as well as faster release, as compared to the homotypic counterparts. Strengths include the rigorous techniques that were used, including biophysical techniques with transmission electron microscopy and the use of molecular dynamics to delineate the mechanism of PRL and GAL interactions at the atomic level. An additional strength is the novel observation of the unidirectional, heterotypic templating competency of PRL fibril seeds for GAL monomers, inducing GAL fibril formation.
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Reviewer #2 (Public Review):
Research on peptide hormones released from the Pituitary, including Prolactin, has shown that the hormones are stored as functional amyloids. Furthermore, it is well established that Prolactin and Galanin are co-stored in secretory granules of the anterior pituitary until they are released into the bloodstream. However, the mechanism by which hormones are stored and released remains a mystery. This study describes the co-aggregation and functional heterotypic amyloid formation of Prolactin and neuro-peptide Galanin in secretory granules. This study suggests that the Prolactin and Galanin interact with each other at high specificity and form functional amyloids. These functional amyloids are heterotypic. Moreover, they demonstrated that Prolactin-Galanin amyloids can form surface-induced secondary fibrils on …
Reviewer #2 (Public Review):
Research on peptide hormones released from the Pituitary, including Prolactin, has shown that the hormones are stored as functional amyloids. Furthermore, it is well established that Prolactin and Galanin are co-stored in secretory granules of the anterior pituitary until they are released into the bloodstream. However, the mechanism by which hormones are stored and released remains a mystery. This study describes the co-aggregation and functional heterotypic amyloid formation of Prolactin and neuro-peptide Galanin in secretory granules. This study suggests that the Prolactin and Galanin interact with each other at high specificity and form functional amyloids. These functional amyloids are heterotypic. Moreover, they demonstrated that Prolactin-Galanin amyloids can form surface-induced secondary fibrils on the surfaces of others. Galanin forms secondary fibrils on Prolactin seeds and Prolactin does not form secondary fibrils on Galanin seeds, indicating that this process occurs in a highly regulated manner. Additionally, they analysed the release of hormone monomers from amyloids in vitro. They found that Prolactin-Galanin functional amyloids are released faster than amyloids formed by Prolactin or Galanin homotypic fibrils. To understand the interactions between Prolactin and Galanin at the atomic level, they have also performed molecular dynamics simulations and docking studies.
A high point of the study is the identification of Prolactin's capability to cross-seed Galanin. This causes amyloid fibrils to be formed. However, in contrast, the Galanin failed in cross-seed the Prolactin. It emphasizes the specificity and regulation in functional amyloid formation. Additionally, the understanding of the interactions between Prolactin and Galanin at the atomic level from MD simulations strengthens the findings. The results of this study did not confirm the possibility of heteromeric fibril formation.
In this study, the authors succeeded in achieving their goals and their conclusions were backed up by their results.
Undoubtedly, this work will have a significant impact on the field of endocrinology and protein aggregation. By studying secretory granules of the pituitary gland, researchers have successfully stepped one step closer to understanding peptide hormone synthesis and release.
-
Reviewer #3 (Public Review):
Maji and coworkers present a tour de force study of the coaggregation of two hormones, prolactin and galanin. Protein aggregation in vivo is much more of a "messy" affair than in the tidy lab of an eppendorph tube and the authors demonstrate an intimate collaboration between these two hormones in the aggregation process. Their work ranges from IHC of tissue slices over experimental biophysics to computational studies, presented in a clear, user-friendly and illustrative manner and overall the conclusions are sound. I found the cartoon diagrams in various figures particularly helpful and of high quality.
Whether their conclusions can be extended to higher-order complexes between multiple hormones (even closer to real life) is the next question to address - but the mind boggles at the number of possibilities …
Reviewer #3 (Public Review):
Maji and coworkers present a tour de force study of the coaggregation of two hormones, prolactin and galanin. Protein aggregation in vivo is much more of a "messy" affair than in the tidy lab of an eppendorph tube and the authors demonstrate an intimate collaboration between these two hormones in the aggregation process. Their work ranges from IHC of tissue slices over experimental biophysics to computational studies, presented in a clear, user-friendly and illustrative manner and overall the conclusions are sound. I found the cartoon diagrams in various figures particularly helpful and of high quality.
Whether their conclusions can be extended to higher-order complexes between multiple hormones (even closer to real life) is the next question to address - but the mind boggles at the number of possibilities to explore.
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