scholarly journals Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

2020 ◽  
Vol 295 (21) ◽  
pp. 7544-7553 ◽  
Author(s):  
Dexter N. Dean ◽  
Jennifer C. Lee
Keyword(s):  
Alternative Splicing ◽  
Amyloid Fibrils ◽  
Limited Proteolysis ◽  
Amyloid Formation ◽  
Functional Amyloid ◽  
Melanin Biosynthesis ◽  
Functional Amyloids ◽  
Fibril Morphology ◽  
Amyloid Assembly ◽  
Β Sheet

The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar β-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a “nucleator” of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

scholarly journals Maturation of the functional mouse CRES amyloid from globular form

2020 ◽  
Vol 117 (28) ◽  
pp. 16363-16372
Author(s):  
Aveline Hewetson ◽  
Nazmul H. Khan ◽  
Matthew J. Dominguez ◽  
Hoa Quynh Do ◽  
R. E. Kusko ◽  
...  
Keyword(s):  
Functional Amyloid ◽  
Conformational Switch ◽  
X Ray Crystallography ◽  
Functional Amyloids ◽  
Globular Form ◽  
Amyloid Assembly ◽  
Β Sheet ◽  
Oligomeric Forms ◽  
Insight Into

The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming β-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid β-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms.

scholarly journals Inhibition of peptide aggregation by means of enzymatic phosphorylation

2016 ◽  
Vol 12 ◽  
pp. 2462-2470 ◽  
Author(s):  
Kristin Folmert ◽  
Malgorzata Broncel ◽  
Hans v. Berlepsch ◽  
Christopher Hans Ullrich ◽  
Mary-Ann Siegert ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Conformational Transition ◽  
Structural Characteristics ◽  
Random Coil ◽  
Amyloid Formation ◽  
Initial Growth ◽  
Amyloid Fibril Formation ◽  
Dependent Protein ◽  
Enzymatic Phosphorylation ◽  
Β Sheet

As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

scholarly journals Cross-seeding Between the Functional Amyloidogenic CRES and CRES3 Family Members and their Regulation of Aβ Assembly

2020 ◽  
pp. jbc.RA120.015307
Author(s):  
Hoa Quynh Do ◽  
Aveline Hewetson ◽  
Collin G Borcik ◽  
Mary Catherine Hastert ◽  
Sandra Whelly ◽  
...  
Keyword(s):  
Normal Component ◽  
X Ray Diffraction ◽  
Cellular Mechanisms ◽  
Functional Amyloid ◽  
X Ray ◽  
Negative Stain ◽  
Transmission Electron ◽  
Evolutionarily Conserved ◽  
Functional Amyloids ◽  
Amyloid Assembly

Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-β reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest CRES and CRES3 heterooligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aβ by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aβ assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.

scholarly journals The β-cell assassin: IAPP cytotoxicity

2017 ◽  
Vol 59 (3) ◽  
pp. R121-R140 ◽  
Author(s):  
Daniel Raleigh ◽  
Xiaoxue Zhang ◽  
Benoît Hastoy ◽  
Anne Clark
Keyword(s):  
Amyloid Fibrils ◽  
Molecular Conformation ◽  
Cell Protein ◽  
Amyloid Formation ◽  
Β Cell ◽  
Islet Amyloid ◽  
The Unfolded Protein Response ◽  
Β Sheet

Islet amyloid polypeptide (IAPP) forms cytotoxic oligomers and amyloid fibrils in islets in type 2 diabetes (T2DM). The causal factors for amyloid formation are largely unknown. Mechanisms of molecular folding and assembly of human IAPP (hIAPP) into β-sheets, oligomers and fibrils have been assessed by detailed biophysical studies of hIAPP and non-fibrillogenic, rodent IAPP (rIAPP); cytotoxicity is associated with the early phases (oligomers/multimers) of fibrillogenesis. Interaction with synthetic membranes promotes β-sheet assembly possibly via a transient α-helical molecular conformation. Cellular hIAPP cytotoxicity can be activated from intracellular or extracellular sites. In transgenic rodents overexpressing hIAPP, intracellular pro-apoptotic signals can be generated at different points in β-cell protein synthesis. Increased cellular trafficking of proIAPP, failure of the unfolded protein response (UPR) or excess trafficking of misfolded peptide via the degradation pathways can induce apoptosis; these data indicate that defects in intracellular handling of hIAPP can induce cytotoxicity. However, there is no evidence for IAPP overexpression in T2DM. Extracellular amyloidosis is directly related to the degree of β-cell apoptosis in islets in T2DM. IAPP fragments, fibrils and multimers interact with membranes causing disruption in vivo and in vitro. These findings support a role for extracellular IAPP in β-sheet conformation in cytotoxicity. Inhibitors of fibrillogenesis are useful tools to determine the aberrant mechanisms that result in hIAPP molecular refolding and islet amyloidosis. However, currently, their role as therapeutic agents remains uncertain.

scholarly journals Quantitating denaturation by formic acid: Imperfect repeats are essential to the stability of the functional amyloid protein FapC

2020 ◽  
Author(s):  
Line Friis Bakmann Christensen ◽  
Jan Stanislaw Nowak ◽  
Thorbjørn Vincent Sønderby ◽  
Signe Andrea Frank ◽  
Daniel Erik Otzen
Keyword(s):  
Formic Acid ◽  
Mechanical Stability ◽  
Complete Removal ◽  
Amyloid Formation ◽  
Amyloid Protein ◽  
Biological Functions ◽  
Functional Amyloid ◽  
High Concentrations ◽  
Functional Amyloids ◽  
The Stability

ABSTRACTBacterial functional amyloids are evolutionarily optimized to aggregate to help them fulfil their biological functions, e.g. to provide mechanical stability to biofilm. Amyloid is formed in Pseudomonas sp. by the protein FapC which contains 3 imperfect repeats connected by long linkers. Stepwise removal of these repeats slows down aggregation and increases the propensity of amyloids to fragment during the fibrillation process, but how these mechanistic properties link to fibril stability is unclear. Here we address this question. The extreme robustness of functional amyloid makes them resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. To quantify this, we first measured the denaturing potency of FA using 3 small acid-resistant proteins (S6, lysozyme and ubiquitin). This revealed a linear relationship between [FA] and the free energy of unfolding with a slope of mFA, as well as a robust correlation between protein residue size and mFA. We then measured the solubilisation of fibrils formed from different FapC variants (with varying number of repeats) as a function of [FA]. The resulting mFA values revealed a decline in the number of residues driving amyloid formation when at least 2 repeats were deleted. The midpoint of denaturation declined monotonically with progressive removal of repeats and correlated with solubility in SDS. Complete removal of all repeats led to fibrils which were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one imperfect repeat is required for the stability of functional amyloid.

scholarly journals Modulation of amyloid assembly by glycosaminoglycans: from mechanism to biological significance

2017 ◽  
Vol 95 (3) ◽  
pp. 329-337 ◽  
Author(s):  
Noé Quittot ◽  
Mathew Sebastiao ◽  
Steve Bourgault
Keyword(s):  
Self Assembly ◽  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Biological Significance ◽  
Active Research ◽  
Living Organisms ◽  
Functional Amyloids ◽  
Almost All ◽  
Misfolding Diseases ◽  
Amyloid Assembly

Glycosaminoglycans (GAGs) are long and unbranched polysaccharides that are abundant in the extracellular matrix and basement membrane of multicellular organisms. These linear polyanionic macromolecules are involved in many physiological functions from cell adhesion to cellular signaling. Interestingly, amyloid fibrils extracted from patients afflicted with protein misfolding diseases are virtually always associated with GAGs. Amyloid fibrils are highly organized nanostructures that have been historically associated with pathological states, such as Alzheimer’s disease and systemic amyloidoses. However, recent studies have identified functional amyloids that accomplish crucial physiological roles in almost all living organisms, from bacteria to insects and mammals. Over the last 2 decades, numerous reports have revealed that sulfated GAGs accelerate and (or) promote the self-assembly of a large diversity of proteins, both inherently amyloidogenic and non-aggregation prone. Despite the fact that many studies have investigated the molecular mechanism(s) by which GAGs induce amyloid assembly, the mechanistic elucidation of GAG-mediated amyloidogenesis still remains the subject of active research. In this review, we expose the contribution of GAGs in amyloid assembly, and we discuss the pathophysiological and functional significance of GAG-mediated fibrillization. Finally, we propose mechanistic models of the unique and potent ability of sulfated GAGs to hasten amyloid fibril formation.

scholarly journals Physical Determinants of Amyloid Assembly in Biofilm Formation

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Maria Andreasen ◽  
Georg Meisl ◽  
Jonathan D. Taylor ◽  
Thomas C. T. Michaels ◽  
Aviad Levin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Self Assembly ◽  
Biofilm Formation ◽  
Amyloid Fibrils ◽  
Environmental Stresses ◽  
Fibril Formation ◽  
Functional Amyloid ◽  
Content Type ◽  
Wide Range ◽  
Amyloid Assembly

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.

scholarly journals Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids

2021 ◽  
Author(s):  
D. Chatterjee ◽  
R.S. Jacob ◽  
S. Ray ◽  
A. Navalkar ◽  
N. Singh ◽  
...  
Keyword(s):  
Neurological Disorders ◽  
Secretory Granules ◽  
Amyloid Formation ◽  
Female Rat ◽  
Secondary Nucleation ◽  
Functional Amyloid ◽  
Rapid Release ◽  
Efficient Storage ◽  
Human Prolactin ◽  
Functional Amyloids

AbstractSynergistic-aggregation and cross-seeding by two different amyloid proteins/peptides are well evident in various neurological disorders. However, this phenomenon is not well studied in functional amyloid aggregation. Here, we show Prolactin (PRL) is associated with lactation in mammals and neuropeptide galanin (GAL), which are co-stored in the lactotrophs facilitates the synergic aggregation in the absence of secretory granules helper molecules glycosaminoglycans (GAGS). Interestingly, although each partner possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation can be mediated by PRL seeds. The specificity of co-aggregation by PRL and GAL along with unidirectional cross-seeding suggests tight regulation of functional amyloid formation during co-storage of these hormones in secretory granule biogenesis of female rat lactotrophs. Further mixed fibrils release the constituent functional hormone much faster than the corresponding individual amyloid formed in presence of GAGs, suggesting that co-aggregation of functionally distant hormones might have evolved for efficient storage, synergistic and rapid release of both hormones upon stimulation. The co-aggregation and cross seeding by two different hormones of completely different structures and sequences (PRL and GAL) suggest a novel mechanism of heterologous amyloid formation both in disease and functional amyloids.

scholarly journals Binding site asymmetry in human transthyretin: insights from a joint neutron and X-ray crystallographic analysis using perdeuterated protein

IUCrJ ◽  
2014 ◽  
Vol 1 (6) ◽  
pp. 429-438 ◽  
Author(s):  
Melina Haupt ◽  
Matthew P. Blakeley ◽  
Stuart J. Fisher ◽  
Sax A. Mason ◽  
Jon B. Cooper ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Crystallographic Analysis ◽  
Amyloid Formation ◽  
Hydrogen Atoms ◽  
New Information ◽  
Binding Pockets ◽  
Dimeric Form ◽  
The Stability ◽  
Single Water Molecule ◽  
Β Sheet

Human transthyretin has an intrinsic tendency to form amyloid fibrils and is heavily implicated in senile systemic amyloidosis. Here, detailed neutron structural studies of perdeuterated transthyretin are described. The analyses, which fully exploit the enhanced visibility of isotopically replaced hydrogen atoms, yield new information on the stability of the protein and the possible mechanisms of amyloid formation. Residue Ser117 may play a pivotal role in that a single water molecule is closely associated with the γ-hydrogen atoms in one of the binding pockets, and could be important in determining which of the two sites is available to the substrate. The hydrogen-bond network at the monomer–monomer interface is more extensive than that at the dimer–dimer interface. Additionally, the edge strands of the primary dimer are seen to be favourable for continuation of the β-sheet and the formation of an extended cross-β structure through sequential dimer couplings. It is argued that the precursor to fibril formation is the dimeric form of the protein.

scholarly journals Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16INK4A

2019 ◽  
Author(s):  
Christoph Göbl ◽  
Vanessa K Morris ◽  
Loes van Dam ◽  
Marieke Visscher ◽  
Paulien E. Polderman ◽  
...  
Keyword(s):  
Redox State ◽  
Amyloid Fibrils ◽  
Structural Rearrangement ◽  
Amyloid Formation ◽  
Cysteine Oxidation ◽  
Cellular Redox ◽  
Cycle Arrest ◽  
Amyloid Fibril Formation ◽  
Helical Protein ◽  
Β Sheet

AbstractAccumulation of the CDK4/6 inhibitor p16INK4A in response to oncogenic transformation leads to cell cycle arrest and senescence and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress and thus could potentially be regulated by the reversible oxidation of cysteines (redox signaling). Indeed, oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all alpha-helical protein, but here we report that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a CDK4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils.

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