Droplet and fibril formation of the functional amyloid Orb2

ABSTRACTA wide range of bacterial pathogens have been shown to form biofilms, which significantly increase their resistance to environmental stresses, such as antibiotics, and are thus of central importance in the context of bacterial diseases. One of the major structural components of these bacterial biofilms are amyloid fibrils, yet the mechanism of fibril assembly and its importance for biofilm formation are currently not fully understood. By studying fibril formationin vitro, in a model system of two common but unrelated biofilm-forming proteins, FapC fromPseudomonas fluorescensand CsgA fromEscherichia coli, we found that the two proteins have a common aggregation mechanism. In both systems, fibril formation proceeds via nucleated growth of linear fibrils exhibiting similar measured rates of elongation, with negligible fibril self-replication. These similarities between two unrelated systems suggest that convergent evolution plays a key role in tuning the assembly kinetics of functional amyloid fibrils and indicates that only a narrow window of mechanisms and assembly rates allows for successful biofilm formation. Thus, the amyloid assembly reaction is likely to represent a means for controlling biofilm formation, both by the organism and by possible inhibitory drugs.IMPORTANCEBiofilms are generated by bacteria, embedded in the formed extracellular matrix. The biofilm's function is to improve the survival of a bacterial colony through, for example, increased resistance to antibiotics or other environmental stresses. Proteins secreted by the bacteria act as a major structural component of this extracellular matrix, as they self-assemble into highly stable amyloid fibrils, making the biofilm very difficult to degrade by physical and chemical means once formed. By studying the self-assembly mechanism of the fibrils from their monomeric precursors in two unrelated bacteria, our experimental and theoretical approaches shed light on the mechanism of functional amyloid assembly in the context of biofilm formation. Our results suggest that fibril formation may be a rate-limiting step in biofilm formation, which in turn has implications on the protein self-assembly reaction as a target for potential antibiotic drugs.

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Molecular Origin of pH-Dependent Fibril Formation of a Functional Amyloid

ChemBioChem ◽

10.1002/cbic.201402074 ◽

2014 ◽

Vol 15 (11) ◽

pp. 1569-1572 ◽

Cited By ~ 20

Author(s):

Ryan P. McGlinchey ◽

Zhiping Jiang ◽

Jennifer C. Lee

Keyword(s):

Fibril Formation ◽

Functional Amyloid ◽

Ph Dependent ◽

Molecular Origin

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Defining an amyloid link Between Parkinson’s disease and melanoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009702117 ◽

2020 ◽

Vol 117 (37) ◽

pp. 22671-22673 ◽

Cited By ~ 1

Author(s):

Dexter N. Dean ◽

Jennifer C. Lee

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein A ◽

Fibril Formation ◽

Human Melanoma ◽

Melanin Content ◽

Functional Amyloid ◽

Multiple Generations ◽

Repeat Domain

An epidemiological connection exists between Parkinson’s disease (PD) and melanoma. α-Synuclein (α-syn), the hallmark pathological amyloid observed in PD, is also elevated in melanoma, where its expression is inversely correlated with melanin content. We present a hypothesis that there is an amyloid link between α-syn and Pmel17 (premelanosomal protein), a functional amyloid that promotes melanogenesis. Using SK-MEL 28 human melanoma cells, we show that endogenous α-syn is present in melanosomes, the organelle where melanin polymerization occurs. Using in vitro cross-seeding experiments, we show that α-syn fibrils stimulate the aggregation of a Pmel17 fragment constituting the repeat domain (RPT), an amyloidogenic domain essential for fibril formation in melanosomes. The cross-seeded fibrils exhibited α-syn−like ultrastructural features that could be faithfully propagated over multiple generations. This cross-seeding was unidirectional, as RPT fibrils did not influence α-syn aggregation. These results support our hypothesis that α-syn, a pathogenic amyloid, modulates Pmel17 aggregation in the melanosome, defining a molecular link between PD and melanoma.

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Droplet and fibril formation of the functional amyloid Orb2

10.1101/2021.02.19.430336 ◽

2021 ◽

Author(s):

Kidist Ashami ◽

Alexander S. Falk ◽

Connor Hurd ◽

Samridhi Garg ◽

Silvia A. Cervantes ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Rna Binding ◽

Rna Binding Proteins ◽

Droplet Formation ◽

Fibril Formation ◽

Long Term Memory ◽

Functional Amyloid

AbstractThe functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low complexity sequences similar to many RNA binding proteins shown to form protein droplets via liquid-liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. We show that soluble Orb2 can indeed phase separate into protein droplets. However, these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. For Orb2 fragments that lack the C-terminal RNA binding domain (RBD), droplet formation is a prerequisite for fibril formation of an otherwise stable monomeric Orb2 solution. Solid-state NMR shows that these fibrils have additional well ordered static domains beside the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into cross-β fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils.

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Immunocytochemical Localization of Amyloid Protein AA in the “Dense Fibrillar Inclusions” of the Reticuloendothelical Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079632 ◽

1977 ◽

Vol 35 ◽

pp. 438-439

Author(s):

T. Shirahama ◽

M. Skinner ◽

A.S. Cohen

Keyword(s):

Amyloid Fibril ◽

Close Association ◽

Gel Filtration ◽

Fibril Formation ◽

Amyloid Protein ◽

Electron Microscopic ◽

Amyloid Deposits ◽

Amyloid Fibril Formation ◽

Electron Microscopic Technique ◽

Lysosomal System

A1thought the mechanisms of amyloidogenesis have not been entirely clarified, proteolysis of the parent proteins may be one of the important steps in the amyloid fibril formation. Recently, we reported that "dense fibrillar inclusions" (DFI), which had the characteristics of lysosomes and contained organized fibrillar profiles as well, were observed in the reticuloendothelial cells in close association with the foci of new amyloid deposits. We considered the findings as evidence for the involvement of lysosomal system in amyloid fibril formation (l). In the present study, we attempted to determine the identity of the contents of the DFI by the use of antisera against the amyloid protein (AA) and an immuno-electron microscopic technique.Amyloidosis was induced in CBA/J mice by daily injections of casein (l). AA was isolated from amyloid-laden spleens by gel filtration and antibody to it was produced in rabbits (2). For immunocytochemistry, the unlabeled antibody enzyme method (3) was employed.

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Ultrastructural analysis of collagen formation in the developing primate amnion

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158078 ◽

1990 ◽

Vol 48 (3) ◽

pp. 106-107

Author(s):

Barry F. King ◽

Grete N. Fry

Keyword(s):

Golgi Apparatus ◽

Collagen Fibril ◽

Fibril Formation ◽

Amniotic Cavity ◽

Cytological Evidence ◽

Mammalian Embryo ◽

Intracellular Location ◽

Collagen Formation ◽

Amniotic Epithelium ◽

Extraembryonic Mesoderm

The amnion surrounding the mammalian embryo consists of the amniotic epithelium facing the amniotic cavity, a layer of extraembryonic mesoderm bordering the exocoelom and an intervening layer of extracellular matrix (Fig. 1). During gestation the amnion expands remarkably to acommodate the rapidly growing embryo. In this study we have examined the process of collagen fibril formation in the developing amnion of the rhesus monkey between 20 and 60 days of gestation.Most cytological evidence of collagen fibril formation was observed in association with the extraembryonic mesodermal cells rather than the amniotic epithelium. The mesodermal cells h ad abundant cisternae of rough endoplasmic reticulum and a prominent Golgi apparatus. Elongated secretory vacuoles were associated with the Golgi apparatus and often contained parallel aggregates of fine filaments (Fig. 2). In some secretory vacuoles, periodic densities also were observed. Some striated collagen fibrils were observed in an apparent intracellular location in long, membrane-limited compartments (Fig. 3). Still other striated fibrils were observed in dense bodies, presumably lysosomes (Fig. 4).

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Functional amyloid: widespread in Nature, diverse in purpose

Essays in Biochemistry ◽

10.1042/bse0560207 ◽

2014 ◽

Vol 56 ◽

pp. 207-219 ◽

Cited By ~ 77

Author(s):

Chi L.L. Pham ◽

Ann H. Kwan ◽

Margaret Sunde

Keyword(s):

Spider Silk ◽

Epigenetic Inheritance ◽

Fibrillar Structure ◽

Cytoplasmic Polyadenylation ◽

Functional Amyloid ◽

Interacting Protein ◽

Diverse Range ◽

Element Binding Protein ◽

Functional Amyloids ◽

Micro Organisms

Amyloids are insoluble fibrillar protein deposits with an underlying cross-β structure initially discovered in the context of human diseases. However, it is now clear that the same fibrillar structure is used by many organisms, from bacteria to humans, in order to achieve a diverse range of biological functions. These functions include structure and protection (e.g. curli and chorion proteins, and insect and spider silk proteins), aiding interface transitions and cell–cell recognition (e.g. chaplins, rodlins and hydrophobins), protein control and storage (e.g. Microcin E492, modulins and PMEL), and epigenetic inheritance and memory [e.g. Sup35, Ure2p, HET-s and CPEB (cytoplasmic polyadenylation element-binding protein)]. As more examples of functional amyloid come to light, the list of roles associated with functional amyloids has continued to expand. More recently, amyloids have also been implicated in signal transduction [e.g. RIP1/RIP3 (receptor-interacting protein)] and perhaps in host defence [e.g. aDrs (anionic dermaseptin) peptide]. The present chapter discusses in detail functional amyloids that are used in Nature by micro-organisms, non-mammalian animals and mammals, including the biological roles that they play, their molecular composition and how they assemble, as well as the coping strategies that organisms have evolved to avoid the potential toxicity of functional amyloid.

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Heparin induces amyloid formation in cultured human isletss

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2870 ◽

2007 ◽

Vol 30 (4) ◽

pp. 92 ◽

Cited By ~ 2

Author(s):

K Potter ◽

K Park

Keyword(s):

Cell Death ◽

B Cells ◽

Islet Transplantation ◽

Islet Cell ◽

Human Islets ◽

Fibril Formation ◽

Amyloid Formation ◽

Secretory Product ◽

Islet Amyloid ◽

Heparin Sulfate

Background: Pancreatic islet transplantation offers improved glycemic control in type 1 diabetic patients above standard insulin therapy, ideally minimizing macro- and microvascular complications of diabetes mellitus. However success is limited thus far, with fewer than 10% of patients retaining insulin independence at two years post-transplantation. In addition to immune rejection, many non-immune factors may promote long-term graft secretory dysfunction and loss of viable graft mass. One such important non-immune factor may be the formation of islet amyloid, a pathologic lesion of the islet in type 2 diabetes that contributes to the progressive loss of b cells in that disease and that has been shown to form rapidly in human islets transplanted into NOD.scid mice. Amyloid deposits are composed primarily of the b cell secretory product islet amyloid polypeptide (IAPP), are cytotoxic, and develop in environments in which b cells are stressed. Heparin sulfate is used as an anti-coagulant in clinical islet transplantation and to prevent the instant blood-mediated inflammatory reaction (IBMIR), which occurs upon contact between islets and blood and may destroy a substantial proportion of the grafted islet mass. However, heparin is also known to stimulate amyloid fibril formation. Methods: To determine whether heparin may enhance amyloid formation in human islets and contribute to graft failure, we cultured isolated human islets in the presence or absence of heparin sulfate (42 and 420 units/ml) for 2 weeks in 11.1 mM glucose. Results: Histological assessment of sections of cultured islets for the presence of amyloid (by thioflavin S staining) revealed a marked, concentration-dependent increase in amyloid deposition following culture in the presence of heparin. Quantitative analysis of these sections showed that the proportion of islet area comprised of amyloid was increased approximately 2-fold (0.15%±0.12% vs 0.46%±0.15% of islet area) following culture in 42 units/ml heparin, and the proportion of islets in which amyloid was detectable (amyloid prevalence) was also increased (35%±24% vs 68%±10% of islets). At 420 units/ml heparin, the amyloid area was even greater (0.23%±0.15% vs 0.97%±0.42% of islet area) as was the amyloid prevalence (53%±29% vs 81%±14% of islets). To affirm that heparin can stimulate IAPP fibrillogenesis and enhance IAPP toxicity, we incubated synthetic human IAPP in the presence of heparin and measured amyloid formation in real time by thioflavin T fluorescence, and cell toxicity by Alamar blue viability assay in transformed rat (INS-1) ß-cell cultures. Heparin stimulated IAPP fibril formation and increased death of INS-1 cells exposed to IAPP (78.2%±10.9% vs 51.8%±12.2% of control viability), suggesting that heparin stimulates IAPP aggregation and toxicity. Remarkably, preliminary assessment of human islets cultured in heparin did not show increased islet cell death by TUNEL staining or loss of insulin immunostaining. Conclusion: In summary, heparin increases amyloid formation in cultured human islets. Although our preliminary data does not suggest that heparin-induced amyloid formation contributes to islet cell death, we speculate that heparin-induced amyloid formation may contribute to graft dysfunction and that caution should be used in the clinical application of this drug in islet transplantation.

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