Functional Amyloids As Natural Storage of Peptide Hormones in Pituitary Secretory Granules

Abstract

Amyloids are highly organized cross β-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer's disease and type II diabetes. However, amyloids may also have a normal biological function as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. Here, we show that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross β-sheet-rich conformation. Thus, in contrast to the original association of amyloids with diseases, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.Cells transport newly synthesized secretory proteins and peptides in vesicles via the endoplasmic reticulum (ER) and Golgi for release into the extracellular space (1,2). Some secretory cells, such as neuroendocrine cells and exocrine cells, store secretory proteins and peptides for extended time periods in a highly concentrated form in membrane-enclosed electron-dense cores termed "secretory granules" (1,3,4), which are derived from the Golgi complex. The dense cores of these granules are made up of large, insoluble secretory protein and peptide aggregates that are formed by self-association (4-6). The granules are not amorphous, but possess a distinct molecular organization, possibly of crystalline structures (7) or large intermolecular aggregates (5,8).Amyloid fibrils are cross-β-sheet structures that are primarily associated with several neurodegenerative diseases including Alzheimer's disease. However, amyloid fibril formation also provides biologically functional entities termed functional amyloids (9) and are present in Escherichia coli (10), silkworm (11), fungi (12), and mammalian skin (13). The cross-β-sheet motif is composed of intermolecular β-sheets along the fibril axis with the β-strands aligned perpendicularly to the fibril axis. An amyloid-like structure of peptide and protein hormones in secretory granules could explain most of their properties.To address the question whether peptide and protein hormones are stored in secretory granules in an amyloid-like aggregation state, we first asked if a diverse set of peptide and protein hormones could form amyloids in vitro at granule-relevant pH 5.5. 42 peptide and protein hormones from multiple species and organs were selected randomly, some linear and some cyclic, with a variety of different three dimensional structures (Table S2). This set of hormones was assayed for a capacity to form amyloids by the amyloid-specific dyes thioflavin T (Thio T), congo red (CR), luminescent conjugated polyelectrolyte probes (LCP), by the conformational transition into β-sheet-rich structure measured by circular dichroism (CD), and by the presence of fibrils in electron microscopy (EM) images. Furthermore, x-ray fiber diffraction was measured for a subset of hormones (Table S1). Only 10 hormones out of the 42 showed significant formation of...

Protein folding: The dark side of proteins

“…Eisenberg, Dobson and others have speculated that the self-complementary stickiness of these short segments might have made them useful building blocks in the earliest stages of life on Earth. Moreover, reports have started to emerge of proteins that function normally in the amyloid state, for example some pituitary hormones 6 . "We know by now of over two dozen native amyloids, so this state is clearly used by biology in a functional way as well as a dysfunctional way, " says Eisenberg.…”

Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen

“…Accumulating evidence, however, has established that some proteins self-assemble into amyloid fibrils that carry out biological roles in the absence of pathology and are known as functional amyloids (Fowler et al, 2007;Pham et al, 2014). In mammals, these include premelanosome protein (PMEL), whose amyloid functions as a scaffold for the synthesis of melanin, several hormones that are stored as amyloids in the pituitary gland, RIP1 and RIP3 (receptor-interacting proteins 1 and 3) amyloids involved in programmed necrosis and α-defensin that forms an amyloid net in the gut mucosa for trapping bacteria (Fowler et al, 2006;Maji et al, 2009;Chu et al, 2012;Li et al, 2012). In addition to our studies of CRES amyloid in the epididymal lumen, we have previously shown that the mouse egg zona pellucida is an amyloid, as is part of the mouse sperm acrosomal matrix, demonstrating that functional amyloids are a normal component of the reproductive tract with integral roles in sperm maturation and fertilization (Guyonnet et al, 2014;Egge et al, 2015).…”

Production of functional inclusion bodies in endotoxin-free Escherichia coli

“…These protein particles, ranging from 50 to around 500 nm in diameter, are emerging protein materials with intriguing biomedical applications Villaverde et al 2012;Villaverde 2012), that can be regarded as biomimetics of functional amyloids (protein repositories for sustained release) acting in the endocrine system (Maji et al 2009;Badtke et al 2009;Mankar et al 2011;Cano-Garrido et al 2013). …”