Direct proof of the amyloid nature of yeast prions [PSI+] and [PIN+] by the method of immunoprecipitation of native fibrils

ABSTRACT Prions are proteins that can exist in several structurally and functionally distinct states, one or more of which is transmissible. Yeast proteins Sup35 and Rnq1 in prion state ([PSI+] and [PIN+], respectively) form oligomers and aggregates, which are transmitted from parents to offspring in a series of generations. Several pieces of indirect evidence indicate that these aggregates also possess amyloid properties, but their binding to amyloid-specific dyes has not been shown in vivo. Meanwhile, it is the specific binding to the Congo Red dye and birefringence in polarized light after such staining that is considered the gold standard for proving the amyloid properties of a protein. Here, we used immunoprecipitation to extract native fibrils of the Sup35 and Rnq1 proteins from yeast strains with different prion status. These fibrils are detected by electron microscopy, stained with Congo Red and exhibit yellow-green birefringence after such staining. All these data show that the Sup35 and Rnq1 proteins in prion state form amyloid fibrils in vivo. The technology of fibrils extraction in combination with standard cytological methods can be used to identify new pathological and functional amyloids in any organism and to analyze the structural features of native amyloid fibrils.

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Studies with Radiolabelled Serum Amyloid P Component Provide Evidence for Turnover and Regression of Amyloid Deposits In Vivo

Clinical Science ◽

10.1042/cs0870289 ◽

1994 ◽

Vol 87 (3) ◽

pp. 289-295 ◽

Cited By ~ 53

Author(s):

Philip N. Hawkins

Keyword(s):

Amyloid Fibrils ◽

Specific Binding ◽

Serum Amyloid ◽

Poor Correlation ◽

Amyloid P Component ◽

Serum Amyloid P ◽

Serum Amyloid P Component ◽

Amyloid Deposits ◽

Amyloid P

1. Quantitative scintigraphic and turnover studies, utilizing the specific binding affinity of serum amyloid P component for amyloid fibrils, have been developed as a tool for evaluating amyloid deposits in vivo. 2. Serial studies in over 300 patients have shown characteristic, diagnostic tissue distributions of amyloid in different types of amyloidosis. There is generally a poor correlation between quantity of amyloid and associated organ dysfunction. 3. Contrary to previous expectations, regression of amyloid has been demonstrated systematically for the first time: AA, AL and variant transthyretin-associated amyloid deposits often regress rapidly, and sometimes completely, if the supply of fibril protein precursors is substantially reduced.

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Protein Co-Aggregation Related to Amyloids: Methods of Investigation, Diversity, and Classification

International Journal of Molecular Sciences ◽

10.3390/ijms19082292 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2292 ◽

Cited By ~ 7

Author(s):

Stanislav Bondarev ◽

Kirill Antonets ◽

Andrey Kajava ◽

Anton Nizhnikov ◽

Galina Zhouravleva

Keyword(s):

Binding Site ◽

Molecular Mechanisms ◽

Amyloid Fibrils ◽

Specific Binding ◽

Protein S ◽

Functional Protein ◽

Functional Roles ◽

Protein Fibrils ◽

Protein Functions ◽

Functional Amyloids

Amyloids are unbranched protein fibrils with a characteristic spatial structure. Although the amyloids were first described as protein deposits that are associated with the diseases, today it is becoming clear that these protein fibrils play multiple biological roles that are essential for different organisms, from archaea and bacteria to humans. The appearance of amyloid, first of all, causes changes in the intracellular quantity of the corresponding soluble protein(s), and at the same time the aggregate can include other proteins due to different molecular mechanisms. The co-aggregation may have different consequences even though usually this process leads to the depletion of a functional protein that may be associated with different diseases. The protein co-aggregation that is related to functional amyloids may mediate important biological processes and change of protein functions. In this review, we survey the known examples of the amyloid-related co-aggregation of proteins, discuss their pathogenic and functional roles, and analyze methods of their studies from bacteria and yeast to mammals. Such analysis allow for us to propose the following co-aggregation classes: (i) titration: deposition of soluble proteins on the amyloids formed by their functional partners, with such interactions mediated by a specific binding site; (ii) sequestration: interaction of amyloids with certain proteins lacking a specific binding site; (iii) axial co-aggregation of different proteins within the same amyloid fibril; and, (iv) lateral co-aggregation of amyloid fibrils, each formed by different proteins.

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Amyloidosis

Oxford Textbook of Medicine ◽

10.1093/med/9780198746690.003.0241 ◽

2020 ◽

pp. 2218-2234

Author(s):

Mark B. Pepys ◽

Philip N. Hawkins

Keyword(s):

Amyloid Fibrils ◽

Amyloid Fibril ◽

Polarized Light ◽

Serum Amyloid ◽

Amyloid P Component ◽

Serum Amyloid P ◽

Serum Amyloid P Component ◽

Amyloid Deposits ◽

Amyloid P

Amyloidosis is the clinical condition caused by extracellular deposition of amyloid in the tissues. Amyloid deposits are composed of amyloid fibrils, abnormal insoluble protein fibres formed by misfolding of their normally soluble precursors. About 30 different proteins can form clinically or pathologically significant amyloid fibrils in vivo as a result of either acquired or hereditary abnormalities. Small, focal, clinically silent amyloid deposits in the brain, heart, seminal vesicles, and joints are a universal accompaniment of ageing. Clinically important amyloid deposits usually accumulate progressively, disrupting the structure and function of affected tissues and lead inexorably to organ failure and death. There is no licensed treatment which can specifically clear amyloid deposits, but intervention which reduces the availability of the amyloid fibril precursor proteins can arrest amyloid accumulation and may lead to amyloid regression with clinical benefit. Pathology—amyloid fibrils bind Congo red dye producing pathognomonic green birefringence when viewed in high-intensity cross-polarized light, and the protein type can be identified by immunostaining or proteomic analysis. Amyloid deposits always contain a nonfibrillar plasma glycoprotein, serum amyloid P component, the universal presence of which is the basis for use of radioisotope-labelled serum amyloid P component as a diagnostic tracer. Clinicopathological correlation—amyloid may be deposited in any tissue of the body, including blood vessels walls and connective tissue matrix; clinical manifestations are correspondingly diverse. Identification of the amyloid fibril protein is always essential for appropriate clinical management. The specific types of amyloidosis covered in this chapter are reactive systemic (AA) amyloidosis, monoclonal immunoglobulin light chain (AL) amyloidosis, and hereditary systemic amyloidoses (including familial amyloid polyneuropathy).

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Calcium-Mediated Signal Transduction during Leaf Senescence

10.32747/1995.7604925.bard ◽

1995 ◽

Author(s):

Sonia Philosoph-Hadas ◽

Richard Crain ◽

Shimon Meir ◽

Nehemia Aharoni ◽

Susan Lurie

Keyword(s):

Signal Transduction ◽

Leaf Senescence ◽

General Pattern ◽

Direct Proof ◽

Indirect Evidence ◽

Early Event ◽

Mesophyll Cells ◽

Detached Leaves

We have examined the possibility that modulation of [Ca2+]cyt may represent a signal which induces senescence processes in leaves, through triggering of lipid hydrolysis leading to the cascade of detriorative events. Characterization of the signal transduction components operating during leaf senescence was gained by studying various Ca2+-dependent activities of parsley and chrysanthemum leaves, in relation to several senescence functions, and in response to senescence-modulating hormones (ethylene,ABA, BA and IAA). Some innovative findings regarding the control of senescence processes by [Ca2+]cyt were established: Several Ca2+-or CaM-related compounds were shown to modulate [Ca2+]cyt and action, thereby affecting whole leaf senescence. The involvement of [Ca2+]cyt in mediating the effects of senescence-modulating hormones has been demonstrated. Loss of energized Ca2+-transport capability of PM was found to an early event in leaf senescence, which occurs before changes in senescence parameters are observed, and while other PM ATPase enzymes still retain about 50% of their activities. A general pattern of increased phosphorylation of PM proteins with advanced senescence, which could be modified by plant hormones applied in vivo (BA) or in vitro (ABA), sa found. Taken together, all this indirect evidence indicate that [Ca2+]cyt is elevated due to the senescence-induced decrease in the ability to extrude Ca2+, which results particularly from reduced PM Ca2++-transport capability rather than increased operation of Ca2+ channels or elevated Ins(1,4,5)P3 levels. The direct proof for such a senescence-related elevation in [Ca2+]cyt was provided for the first time by the Ca2+ imaging measures with fura-2, showing a rise in [Ca2+]cyt of mesophyll cells upon senescence induction, which preceeded changes in typical senescence characteristics. This research provides strong evidence for regarding the rise in [Ca2+]cyt as a primary event in induction of the senescence syndrome in detached leaves. The findings have also broad implications for postharvest handling of leafy crops and ornamentals, and open new avenues for employing Ca2+-related inhibitors to delay leaf senescence.

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Fluorescence detection of amyloid deposits in human tissues using histochemical dyes

Bulletin of Russian State Medical University ◽

10.24075/brsmu.2021.034 ◽

2021 ◽

Author(s):

VV Guselnikova ◽

DA Sufieva ◽

DL Tsyba ◽

DE Korzhevskii

Keyword(s):

Fluorescence Microscopy ◽

Fluorescence Detection ◽

Congo Red ◽

Amyloid Fibrils ◽

Polarized Light ◽

Methyl Violet ◽

Human Tissues ◽

Transthyretin Amyloidosis ◽

Amyloid Deposits ◽

Β Amyloid

Recently, fluorescence microscopy becomes more available, presenting new opportunities to face several challenges of experimental biology and medicine. The study was aimed to assess the effectiveness of fluorescence microscopy for the identification of amyloid deposits in human tissues. Post-mortem samples of the myocardium (n = 12) and cerebral cortex (n = 8) obtained from subjects of both sexes aged 60–98 with verified amyloidosis were used as a material for the study. The specimens were stained using 11 different histochemical dyes and subsequently analyzed by light and fluorescence microscopy. Qualitative and quantitative analysis has shown that Thioflavin T is the most effective stain for fluorescence detection of β- and transthyretin amyloid in human tissues. Congo red staining is highly effective for the detection of transthyretin amyloidosis, however, it is ill-suited for the identification of β-amyloid plaques. It has been found that the ability of Congo red to exhibit fluorescence when binding to amyloid fibrils can be used for verification of amyloid deposits instead of the traditional polarized light microscopy. As has been first noted, methyl violet can selectively bind to β-amyloid with fluorescent complex formation. In addition, methyl violet treatment effectively reduces the autofluorescent background in the nervous tissue. This makes methyl violet staining a promising diagnostic tool for Alzheimer's-type pathology.

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A clinicopathological study of primary localised cutaneous amyloidosis

International Journal of Research in Dermatology ◽

10.18203/issn.2455-4529.intjresdermatol20174649 ◽

2017 ◽

Vol 3 (4) ◽

pp. 470 ◽

Cited By ~ 1

Author(s):

Vinitha V. Panicker ◽

Soumya Jagadeesan ◽

Gopikrishnan Anjaneyan ◽

Lekshmi S. ◽

Malini Eapen ◽

...

Keyword(s):

Congo Red ◽

Amyloid Fibrils ◽

Immunofluorescence Microscopy ◽

Cultural Practices ◽

Diagnostic Yield ◽

Polarized Light ◽

Published Data ◽

Polarized Microscopy ◽

Congo Red Staining ◽

Macular Amyloidosis

Background: Amyloidosis is defined as extracellular deposits of heterogenic, misfolded proteins, amyloid fibrils, in various tissues. The term primary cutaneous amyloidosis (PLCA) usually includes macular amyloidosis (MA), lichen amyloidosis (LA) and nodular amyloidosis. Primary cutaneous amyloidosis is very common in Kerala probably due to socio-cultural practices. There has been no published data on PLCA from Kerala thus we undertook this study. The objectives of the study were to correlate clinical features of primary localized cutaneous amyloidosis with histopathologic findings; to evaluate the sensitivity of Congo red staining with polarized light in histopathologically proven primary localized cutaneous amyloidosis)Methods: We undertook an observational analysis for a period of 2 years from May 2012 to April 2014 in the Department of Dermatology, Amrita Institute, Cochin. All cases clinically diagnosed as cutaneous amylodosis were included in the study. After informed consent, skin biopsy was taken. The histopathologic sections were stained with Congo red and seen under polarized microscopy for apple green birefrengance.Results: A total of 70 patients were included in the study. Of the 70 cases, there were 20 males and 50 females. The most common clinical type was lichen amylodosis observed in 32 patients followed by macular amylodosis (28) and biphasic amyloidosis (10) cases. Histopathological compatibility was seen in 71% of MA and 89% cases of LA. Congored positivity was seen in 53.8%. Congored stain under immunofluorescence microscopy was done for 30 patients which gave a positivity of 85% which indicates that it is more sensitive that polarizing microscopy.Conclusions: Our study showed that the most common type is lichen amylodosis. Histopathology and congo red staining with polarized light is a valuable aid in diagnosis. Congo red stain under immunofluorescence microscopy has greater sensitivity and improves the diagnostic yield.

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First Case of Nodular Localized Primary Cutaneous Amyloidosis Treated With Bortezomib and Dexamethasone

Journal of Investigative Medicine High Impact Case Reports ◽

10.1177/23247096211058488 ◽

2021 ◽

Vol 9 ◽

pp. 232470962110584

Author(s):

Noman Ahmed Jang Khan ◽

Emma Nellhaus ◽

Doreen Griswold ◽

Muhammad Omer Jamil

Keyword(s):

Mass Spectrometry ◽

Laser Therapy ◽

Congo Red ◽

Amyloid Fibrils ◽

Laser Microdissection ◽

Polarized Light ◽

Surgical Excision ◽

Rare Form ◽

First Case ◽

Amyloid Deposits

Nodular localized cutaneous amyloidosis is a rare form of cutaneous amyloidosis and is characterized by an extracellular deposition of insoluble amyloid fibrils which are either primarily cutaneous or a manifestation of an underlying systemic amyloidosis. Biopsy of the lesion is mandatory for the diagnosis, and histopathology shows diffuse amyloid deposits with plasmacytic infiltration. Apple-green birefringence characteristic of amyloidosis is observed when stained with Congo red and viewed under polarized light. Amyloid subtyping is done with laser microdissection followed by mass spectrometry. Majority of these lesions do not require any treatment but surgical excision, shave excision, laser therapy, and radiotherapy can be considered for symptomatic nodular localized primary cutaneous amyloidosis (NLPCA). We present a case of recurrent NLPCA in a 64-year-old woman who was treated with bortezomib and dexamethasone after failing several local therapies with excellent response.

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β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms222111316 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11316

Author(s):

Anna I. Sulatskaya ◽

Anastasiia O. Kosolapova ◽

Alexander G. Bobylev ◽

Mikhail V. Belousov ◽

Kirill S. Antonets ◽

...

Keyword(s):

Amyloid Fibrils ◽

Biological Functions ◽

Interspecies Interactions ◽

Structural Relationships ◽

Fibrillar Morphology ◽

Dimeric Form ◽

Functional Amyloids ◽

Partially Unfolded

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another—followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis (“on-pathway state”), or can be formed as a result of an alternative assembly of partially unfolded monomers (“off-pathway state”). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only “the tip of the iceberg”. Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.

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Modulators of amyloid protein aggregation and toxicity: EGCG and CLR01

Translational Neuroscience ◽

10.2478/s13380-013-0137-y ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 17

Author(s):

Aida Attar ◽

Farid Rahimi ◽

Gal Bitan

Keyword(s):

Protein Aggregation ◽

Drug Targets ◽

Amyloid Fibrils ◽

Specific Binding ◽

Amyloid Β ◽

Amyloid Β Protein ◽

Amyloid Protein ◽

Islet Amyloid

AbstractAbnormal protein folding and self-assembly causes over 30 cureless human diseases for which no disease-modifying therapies are available. The common side to all these diseases is formation of aberrant toxic protein oligomers and amyloid fibrils. Both types of assemblies are drug targets, yet each presents major challenges to drug design, discovery, and development. In this review, we focus on two small molecules that inhibit formation of toxic amyloid protein assemblies — the green-tea derivative (−)-epigallocatechin-3-gallate (EGCG), which was identified through a combination of epidemiologic data and a compound library screen, and the molecular tweezer CLR01, whose inhibitory activity was discovered in our group based on rational reasoning, and subsequently confirmed experimentally. Both compounds act in a manner that is not specific to one particular protein and thus are useful against a multitude of amyloidogenic proteins, yet they act via distinct putative mechanisms. CLR01 disrupts protein aggregation through specific binding to lysine residues, whereas the mechanisms underlying the activity of EGCG are only recently beginning to unveil. We discuss current in vitro and, where available, in vivo literature related to EGCG and CLR01’s effects on amyloid β-protein, α-synuclein, transthyretin, islet amyloid polypeptide, and calcitonin. We also describe the toxicity, pharmacokinetics, and mechanism of action of each compound.

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Architectural factor HMGA induces promoter bending and recruits C/EBP and GATA during silkmoth chorion gene regulation

Biochemical Journal ◽

10.1042/bj20081012 ◽

2008 ◽

Vol 416 (1) ◽

pp. 85-97 ◽

Cited By ~ 8

Author(s):

Argyris Papantonis ◽

Josef Vanden Broeck ◽

Rena Lecanidou

Keyword(s):

Gene Regulation ◽

Protein Complexes ◽

Specific Binding ◽

Binding Protein ◽

Specific Interaction ◽

High Mobility ◽

Structural Features ◽

Gene Promoters ◽

Significant Similarity

A protein displaying significant similarity to mammalian HMGA (high-mobility group A) proteins, but also bearing unique structural features, was isolated from silkmoth (Bombyx mori) follicular cells. This factor, named BmHMGA, exhibits specific binding preference for chorion gene promoter elements and induces DNA bending thereon. BmHMGA deploys temporal-specific interaction with transcription factors BmC/EBP (C/EBP is CCAAT/enhancer-binding protein) and BmGATAβ during follicle maturation. The respective protein complexes can be detected on chorion gene promoters in vivo, with different developmental profiles each time. Analogous interaction takes place on the putative promoter of the BmC/EBP gene, hinting towards a transcriptional circuit that is responsible for the progress of choriogenesis as a whole. Finally, transient suppression of BmHMGA expression led to down-regulation of chorion genes and the BmC/EBP gene, and revealed recruitment of BmC/EBP, BmGATAβ and TFIID (transcription factor IID)/TBP (TATA-box-binding protein) by BmHMGA. A revised model for chorion gene regulation is discussed in view of these findings.

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