Publications by authors named "Yury O Chernoff"

72 Publications

Modeling Amyloid Aggregation Kinetics: A Case Study with Sup35NM.

J Phys Chem B 2021 05 7;125(19):4955-4963. Epub 2021 May 7.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States.

Understanding the aggregation mechanism of amyloid proteins, such as Sup35NM, is essential to understanding amyloid diseases. Significant recent work has focused on using the fluorescence of thioflavin T (ThT), which undergoes a red shift when bound to amyloid aggregates, to monitor amyloid fibril formation. In the present study, the progression of the total mass of aggregates during fibril formation is monitored for initial monomer concentrations in order to infer the relevant aggregation mechanisms. This workflow was implemented using the amyloid-forming fragment Sup35NM under different agitation conditions and for initial monomer concentrations spanning 2 orders of magnitude. The analysis suggests that primary nucleation, monomeric elongation, secondary nucleation, and fragmentation might all be relevant, but their relative importance could not be determined unambiguously, despite the large set of high-quality data. Discriminating between the fibril-generating processes is shown to require additional information, such as a fibril length distribution. Using Sup35NM as a case study, a framework for fitting the parameters of arbitrary amyloid aggregation kinetics is developed based on a population balance model (PBM), which resolves not only the total aggregate mass (monitored experimentally via ThT fluorescence) but the entire fibril length distribution over time. In addition to the rich new set of ThT fluorescence data, we have reanalyzed a previously published aggregate size distribution using this method. With the size distribution, it was determined that in the reanalyzed experiment, secondary nucleation generated significantly fewer new Sup35NM fibrils than fragmentation. The proposed strategy of applying the same PBM to a combination of kinetic data from fluorescence monitoring and experimental fibril length distributions will allow the inference of aggregation mechanisms with far greater confidence than fluorescence studies alone.
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http://dx.doi.org/10.1021/acs.jpcb.0c11250DOI Listing
May 2021

Development of molecular tools for diagnosis of Alzheimer's disease that are based on detection of amyloidogenic proteins.

Prion 2021 Dec;15(1):56-69

Laboratory of Amyloid Biology, St. Petersburg State University, St. Petersburg, Russia.

Alzheimer's disease (AD) is the most common form of dementia that usually occurs among older people. AD results from neuronal degeneration that leads to the cognitive impairment and death. AD is incurable, typically develops over the course of many years and is accompanied by a loss of functional autonomy, making a patient completely dependent on family members and/or healthcare workers. Critical features of AD are pathological polymerization of Aβ peptide and microtubule-associated protein tau, accompanied by alterations of their conformations and resulting in accumulation of cross-β fibrils (amyloids) in human brains. AD apparently progresses asymptomatically for years or even decades before the appearance of symptoms. Therefore, development of the early AD diagnosis at a pre-symptomatic stage is essential for potential therapies. This review is focused on current and potential molecular tools (including non-invasive methods) that are based on detection of amyloidogenic proteins and can be applicable to early diagnosis of AD.: Aβ - amyloid-β peptide; AβO - amyloid-β oligomers; AD - Alzheimer's disease; ADRDA - Alzheimer's Disease and Related Disorders Association; APH1 - anterior pharynx defective 1; APP - amyloid precursor protein; BACE1 - β-site APP-cleaving enzyme 1; BBB - brain blood barrier; CJD - Creutzfeldt-Jakob disease; CRM - certified reference material; CSF - cerebrospinal fluid; ELISA - enzyme-linked immunosorbent assay; FGD - F-fluorodesoxyglucose (2-deoxy-2-[F]fluoro-D-glucose); IP-MS - immunoprecipitation-mass spectrometry assay; MCI - mild cognitive impairment; MDS - multimer detection system; MRI - magnetic resonance imaging; NIA-AA - National Institute on Ageing and Alzheimer's Association; NINCDS - National Institute of Neurological and Communicative Disorders and Stroke; PEN2 - presenilin enhancer 2; PET - positron emission tomography; PiB - Pittsburgh Compound B; PiB-SUVR - PIB standardized uptake value ratio; PMCA - Protein Misfolding Cycling Amplification; PrP - Prion Protein; P-tau - hyperphosphorylated tau protein; RMP - reference measurement procedure; RT-QuIC - real-time quaking-induced conversion; SiMoA - single-molecule array; ThT - thioflavin T; TSEs - Transmissible Spongiform Encephslopathies; T-tau - total tau protein.
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http://dx.doi.org/10.1080/19336896.2021.1917289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096329PMC
December 2021

Design and synthesis of novel tacrine-indole hybrids as potential multitarget-directed ligands for the treatment of Alzheimer's disease.

Future Med Chem 2021 May 8;13(9):785-804. Epub 2021 Apr 8.

Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, Hradec Kralove, 500 05, Czech Republic.

The authors report on the synthesis and biological evaluation of new compounds whose structure combines tacrine and indole moieties. Tacrine-indole heterodimers were designed to inhibit cholinesterases and β-amyloid formation, and to cross the blood-brain barrier. The most potent new acetylcholinesterase inhibitors were compounds and (IC = 25 and 39 nM, respectively). Compound displayed considerably higher selectivity for acetylcholinesterase relative to human plasma butyrylcholinesterase in comparison to compound (selectivity index: IC [butyrylcholinesterase]/IC [acetylcholinesterase] = 3 and 0.6, respectively). Furthermore, compound inhibited β-amyloid-dependent amyloid nucleation in the yeast-based prion nucleation assay and displayed no dsDNA destabilizing interactions with DNA. Compounds and displayed a high probability of crossing the blood-brain barrier. The results support the potential of for future development as a dual-acting therapeutic agent in the prevention and/or treatment of Alzheimer's disease.
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http://dx.doi.org/10.4155/fmc-2020-0184DOI Listing
May 2021

Regulation of the endocytosis and prion-chaperoning machineries by yeast E3 ubiquitin ligase Rsp5 as revealed by orthogonal ubiquitin transfer.

Cell Chem Biol 2021 Feb 23. Epub 2021 Feb 23.

Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA. Electronic address:

Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an "orthogonal UB transfer" (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2, which are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.
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http://dx.doi.org/10.1016/j.chembiol.2021.02.005DOI Listing
February 2021

Functional Mammalian Amyloids and Amyloid-Like Proteins.

Life (Basel) 2020 Aug 21;10(9). Epub 2020 Aug 21.

Laboratory of Amyloid Biology, St. Petersburg State University, 199034 St. Petersburg, Russia.

Amyloids are highly ordered fibrous cross-β protein aggregates that are notorious primarily because of association with a variety of incurable human and animal diseases (termed amyloidoses), including Alzheimer's disease (AD), Parkinson's disease (PD), type 2 diabetes (T2D), and prion diseases. Some amyloid-associated diseases, in particular T2D and AD, are widespread and affect hundreds of millions of people all over the world. However, recently it has become evident that many amyloids, termed "functional amyloids," are involved in various activities that are beneficial to organisms. Functional amyloids were discovered in diverse taxa, ranging from bacteria to mammals. These amyloids are involved in vital biological functions such as long-term memory, storage of peptide hormones and scaffolding melanin polymerization in animals, substrate attachment, and biofilm formation in bacteria and fungi, etc. Thus, amyloids undoubtedly are playing important roles in biological and pathological processes. This review is focused on functional amyloids in mammals and summarizes approaches used for identifying new potentially amyloidogenic proteins and domains.
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http://dx.doi.org/10.3390/life10090156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555005PMC
August 2020

Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association.

Int J Mol Sci 2020 Jul 16;21(14). Epub 2020 Jul 16.

School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332-2000, USA.

Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-β ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.
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http://dx.doi.org/10.3390/ijms21145038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403958PMC
July 2020

Application of yeast to studying amyloid and prion diseases.

Adv Genet 2020 4;105:293-380. Epub 2020 May 4.

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, United States.

Amyloids are fibrous cross-β protein aggregates that are capable of proliferation via nucleated polymerization. Amyloid conformation likely represents an ancient protein fold and is linked to various biological or pathological manifestations. Self-perpetuating amyloid-based protein conformers provide a molecular basis for transmissible (infectious or heritable) protein isoforms, termed prions. Amyloids and prions, as well as other types of misfolded aggregated proteins are associated with a variety of devastating mammalian and human diseases, such as Alzheimer's, Parkinson's and Huntington's diseases, transmissible spongiform encephalopathies (TSEs), amyotrophic lateral sclerosis (ALS) and transthyretinopathies. In yeast and fungi, amyloid-based prions control phenotypically detectable heritable traits. Simplicity of cultivation requirements and availability of powerful genetic approaches makes yeast Saccharomyces cerevisiae an excellent model system for studying molecular and cellular mechanisms governing amyloid formation and propagation. Genetic techniques allowing for the expression of mammalian or human amyloidogenic and prionogenic proteins in yeast enable researchers to capitalize on yeast advantages for characterization of the properties of disease-related proteins. Chimeric constructs employing mammalian and human aggregation-prone proteins or domains, fused to fluorophores or to endogenous yeast proteins allow for cytological or phenotypic detection of disease-related protein aggregation in yeast cells. Yeast systems are amenable to high-throughput screening for antagonists of amyloid formation, propagation and/or toxicity. This review summarizes up to date achievements of yeast assays in application to studying mammalian and human disease-related aggregating proteins, and discusses both limitations and further perspectives of yeast-based strategies.
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http://dx.doi.org/10.1016/bs.adgen.2020.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527210PMC
January 2021

Risk of Alzheimer's Disease in Cancer Patients: Analysis of Mortality Data from the US SEER Population-Based Registries.

Cancers (Basel) 2020 Mar 26;12(4). Epub 2020 Mar 26.

School of Biological Sciences, Georgia Institute of Technology, Krone Engineered Biosystems Building, 950 Atlantic Drive NW, Atlanta, GA 30332-2000, USA.

Previous studies have reported an inverse association between cancer and Alzheimer's disease (AD), which are leading causes of human morbidity and mortality. We analyzed the SEER (Surveillance, Epidemiology, and End Results) data to estimate the risk of AD death in (i) cancer patients relative to reference populations stratified on demographic and clinical variables, and (ii) female breast cancer (BC) patients treated with chemotherapy or radiotherapy, relative to those with no/unknown treatment status. Our results demonstrate the impact of race, cancer type, age and time since cancer diagnosis on the risk of AD death in cancer patients. While the risk of AD death was decreased in white patients diagnosed with various cancers at 45 or more years of age, it was increased in black patients diagnosed with cancers before 45 years of age (likely due to early onset AD). Chemotherapy decreased the risk of AD death in white women diagnosed with BC at the age of 65 or more, however radiotherapy displayed a more complex pattern with early decrease and late increase in the risk of AD death during a prolonged time interval after the treatment. Our data point to links between molecular mechanisms involved in cancer and AD, and to the potential applicability of some anti-cancer treatments against AD.
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http://dx.doi.org/10.3390/cancers12040796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226270PMC
March 2020

Protein Misfolding during Pregnancy: New Approaches to Preeclampsia Diagnostics.

Int J Mol Sci 2019 Dec 7;20(24). Epub 2019 Dec 7.

Laboratory of Amyloid Biology, St. Petersburg State University, 199034 St. Petersburg, Russia.

Preeclampsia (PE) is a multisystem heterogeneous complication of pregnancy remaining a leading cause of maternal and perinatal morbidity and mortality over the world. PE has a large spectrum of clinical features and symptoms, which make diagnosis challenging. Despite a long period of studying, PE etiology is still unclear and there are no reliable rapid tests for early diagnosis of this disease. During the last decade, it was shown that proteins misfolding and aggregation are associated with PE. Several proteins, including amyloid beta peptide, transthyretin, alpha-1 antitrypsin, albumin, IgG k-free light chains, and ceruloplasmin are dysregulated in PE, resulting in toxic deposition of amyloid-like aggregates in the placenta and body fluids. It is also possible that aggregated proteins induce defective trophoblast invasion, placental ischemia, ER stress, and promote PE manifestation. The fact that protein aggregation is an emerging biomarker of PE provides an opportunity to develop new diagnostic approaches based on amyloids special features, such as Congo red (CR) staining and thioflavin T (ThT) enhanced fluorescence.
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http://dx.doi.org/10.3390/ijms20246183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941028PMC
December 2019

Yeast Models for Amyloids and Prions: Environmental Modulation and Drug Discovery.

Molecules 2019 Sep 18;24(18). Epub 2019 Sep 18.

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

Amyloids are self-perpetuating protein aggregates causing neurodegenerative diseases in mammals. Prions are transmissible protein isoforms (usually of amyloid nature). Prion features were recently reported for various proteins involved in amyloid and neural inclusion disorders. Heritable yeast prions share molecular properties (and in the case of polyglutamines, amino acid composition) with human disease-related amyloids. Fundamental protein quality control pathways, including chaperones, the ubiquitin proteasome system and autophagy are highly conserved between yeast and human cells. Crucial cellular proteins and conditions influencing amyloids and prions were uncovered in the yeast model. The treatments available for neurodegenerative amyloid-associated diseases are few and their efficiency is limited. Yeast models of amyloid-related neurodegenerative diseases have become powerful tools for high-throughput screening for chemical compounds and FDA-approved drugs that reduce aggregation and toxicity of amyloids. Although some environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. Environmental stresses trigger amyloid formation and loss, acting either via influencing intracellular concentrations of the amyloidogenic proteins or via heterologous inducers of prions. Studies of environmental and physiological regulation of yeast prions open new possibilities for pharmacological intervention and/or prophylactic procedures aiming on common cellular systems rather than the properties of specific amyloids.
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http://dx.doi.org/10.3390/molecules24183388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767215PMC
September 2019

Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Prion by Heat Shock.

Genetics 2019 07 29;212(3):757-771. Epub 2019 May 29.

School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332

Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [ ], a prion isoform of the yeast translation termination factor Sup35 This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [ ] destabilization. Moreover, deletion of the gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [ ], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104-GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [ ], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone.
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http://dx.doi.org/10.1534/genetics.119.302237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614889PMC
July 2019

Biomolecular Assemblies: Moving from Observation to Predictive Design.

Chem Rev 2018 12 3;118(24):11519-11574. Epub 2018 Oct 3.

Department of Chemistry , Emory University , Atlanta , Georgia 30322 , United States.

Biomolecular assembly is a key driving force in nearly all life processes, providing structure, information storage, and communication within cells and at the whole organism level. These assembly processes rely on precise interactions between functional groups on nucleic acids, proteins, carbohydrates, and small molecules, and can be fine-tuned to span a range of time, length, and complexity scales. Recognizing the power of these motifs, researchers have sought to emulate and engineer biomolecular assemblies in the laboratory, with goals ranging from modulating cellular function to the creation of new polymeric materials. In most cases, engineering efforts are inspired or informed by understanding the structure and properties of naturally occurring assemblies, which has in turn fueled the development of predictive models that enable computational design of novel assemblies. This Review will focus on selected examples of protein assemblies, highlighting the story arc from initial discovery of an assembly, through initial engineering attempts, toward the ultimate goal of predictive design. The aim of this Review is to highlight areas where significant progress has been made, as well as to outline remaining challenges, as solving these challenges will be the key that unlocks the full power of biomolecules for advances in technology and medicine.
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http://dx.doi.org/10.1021/acs.chemrev.8b00038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650774PMC
December 2018

A standard model of Alzheimer's disease?

Prion 2018 9;12(5-6):261-265. Epub 2018 Oct 9.

c School of Biological Sciences , Georgia Institute of Technology , Atlanta , GA , USA.

The recent Research Framework proposed by the US National Institute on Aging and the Alzheimer's Association (NIA-AA) recommends that Alzheimer's disease be defined by its specific biology rather than by non-specific neurodegenerative and syndromal features. By affirming markers of abnormal Aβ and tau proteins as the essential pathobiological signature of Alzheimer's disease, the Framework tacitly reinforces the amyloid (Aβ) cascade as the leading theory of Alzheimer pathogenesis. In light of recent evidence that the cascade is driven by the misfolding and templated aggregation of Aβ and tau, we believe that an empirically grounded Standard Model of Alzheimer's pathogenesis is within reach. A Standard Model can clarify and consolidate existing information, contextualize risk factors and the complex disease phenotype, identify testable hypotheses for future research, and pave the most direct path to effective prevention and treatment.
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http://dx.doi.org/10.1080/19336896.2018.1525256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277193PMC
July 2019

Modulation of the Formation of Aβ- and Sup35NM-Based Amyloids by Complex Interplay of Specific and Nonspecific Ion Effects.

J Phys Chem B 2018 05 3;122(19):4972-4981. Epub 2018 May 3.

Laboratory of Amyloid Biology and Institute of Translational Biomedicine , St. Petersburg State University , St. Petersburg 199034 , Russia.

In vitro formation of highly ordered protein aggregates, amyloids, is influenced by the presence of ions. Here, we have studied the effect of anions on amyloid fibril formation by two different amyloidogenic proteins, human amyloid beta-42 (Aβ), associated with Alzheimer disease and produced recombinantly with an N-terminal methionine (Met-Aβ), and histidine-tagged NM fragment of Sup35 protein (Sup35NM-His), a yeast release factor controlling protein-based inheritance, at pH values above and below their isoelectric points. We demonstrate here that pH plays a critical role in determining the effect of ions on the aggregation of Met-Aβ and Sup35NM-His. Further, the electrophoretic mobilities of Met-Aβ and Sup35NM-His were measured in the presence of different anions at pH above and below the isoelectric points to understand how anions interact with these proteins when they bear a net positive or negative charge. We find that although ion-protein interactions generally follow expectations based on the anion positions within the Hofmeister series, there are qualitative differences in the aggregation behavior of Met-Aβ and Sup35NM-His. These differences arise from a competition between nonspecific charge neutralization and screening effects and specific ion adsorption and can be explained by the different biochemical and biophysical properties of Met-Aβ and Sup35NM-His.
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http://dx.doi.org/10.1021/acs.jpcb.7b12836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932987PMC
May 2018

Mammalian amyloidogenic proteins promote prion nucleation in yeast.

J Biol Chem 2018 03 12;293(9):3436-3450. Epub 2018 Jan 12.

From the School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332,

Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-β (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes.
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http://dx.doi.org/10.1074/jbc.M117.809004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836139PMC
March 2018

Proteolysis suppresses spontaneous prion generation in yeast.

J Biol Chem 2017 12 16;292(49):20113-20124. Epub 2017 Oct 16.

Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan. Electronic address:

Prions are infectious proteins that cause fatal neurodegenerative disorders including Creutzfeldt-Jakob and bovine spongiform encephalopathy (mad cow) diseases. The yeast [] prion is formed by the translation-termination factor Sup35, is the best-studied prion, and provides a useful model system for studying such diseases. However, despite recent progress in the understanding of prion diseases, the cellular defense mechanism against prions has not been elucidated. Here, we report that proteolytic cleavage of Sup35 suppresses spontaneous generation of the [] prion. We found that during yeast growth in glucose media, a maximum of 40% of Sup35 is cleaved at its N-terminal prion domain. This cleavage requires the vacuolar proteases PrA-PrB. Cleavage occurs in a manner dependent on translation but independently of autophagy between the glutamine/asparagine-rich (Q/N-rich) stretch critical for prion formation and the oligopeptide-repeat region required for prion maintenance, resulting in the removal of the Q/N-rich stretch from the Sup35 N terminus. The complete inhibition of Sup35 cleavage, by knocking out either PrA (Δ) or PrB (Δ), increased the rate of formation of [] prion up to ∼5-fold, whereas the activation of Sup35 cleavage, by overproducing PrB, inhibited [] formation. On the other hand, activation of the PrB pathway neither cleaved the amyloid conformers of Sup35 in [] strains nor eliminated preexisting []. These findings point to a mechanism antagonizing prion generation in yeast. Our results underscore the usefulness of the yeast [] prion as a model system to investigate defense mechanisms against prion diseases and other amyloidoses.
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http://dx.doi.org/10.1074/jbc.M117.811323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724000PMC
December 2017

Differential effects of chaperones on yeast prions: CURrent view.

Curr Genet 2018 Apr 20;64(2):317-325. Epub 2017 Sep 20.

Department of Genetics and Biotechnology, St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg, 199034, Russia.

Endogenous yeast amyloids that control heritable traits and are frequently used as models for human amyloid diseases are termed yeast prions. Yeast prions, including the best studied ones ([PSI ] and [URE3]), propagate via intimate interactions with molecular chaperones. Different yeast prions exhibit differential responses to changes in levels, functionality or localization of the components of chaperone machinery. Here, we provide additional data confirming differential effects of chaperones (and specifically, Hsp40s) on yeast prions and summarize current knowledge of the mechanisms underlying chaperone specificities. Contrary to frequent statements in literature, overproduction of the Hsp104 chaperone antagonizes both [PSI ] and [URE3] prions, while overproduction of the Hsp70-Ssa1 chaperone antagonizes [URE3] prion only in some, but not in all strains. Recently, we demonstrated that the relocalization of a fraction of the Hsp40 chaperone Sis1 from the cytosol to the nucleus by the chaperone-sorting factor Cur1 exhibits opposite effects on [PSI ] and [URE3] prions. We suggest that the response of prions to changes in Sis1 localization represents a combination of the effects of Sis1 shortage on fragmentation of prion aggregates and on malpartition of prion aggregates during a cell division. Differences in sensitivity of prion fragmentation to Sis1 and in relative inputs of fragmentation and malpartition in prion propagation result in opposite effects of Sis1 relocalization on [PSI ] and [URE3].
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http://dx.doi.org/10.1007/s00294-017-0750-3DOI Listing
April 2018

Prion-based memory of heat stress in yeast.

Prion 2017 05 19;11(3):151-161. Epub 2017 May 19.

a Department of Biochemistry , Emory University School of Medicine , Atlanta , GA , USA.

Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices.  We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses.
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http://dx.doi.org/10.1080/19336896.2017.1328342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480388PMC
May 2017

To CURe or not to CURe? Differential effects of the chaperone sorting factor Cur1 on yeast prions are mediated by the chaperone Sis1.

Mol Microbiol 2017 Jul 9;105(2):242-257. Epub 2017 May 9.

Department of Genetics and Biotechnology, St. Petersburg State University, St. Petersburg 199034, Russia.

Yeast self-perpetuating protein aggregates (prions) provide a convenient model for studying various components of the cellular protein quality control system. Molecular chaperones and chaperone-sorting factors, such as yeast Cur1 protein, play key role in proteostasis via tight control of partitioning and recycling of misfolded proteins. In this study, we show that, despite the previously described ability of Cur1 to antagonize the yeast prion [URE3], it enhances propagation and phenotypic manifestation of another prion, [PSI ]. We demonstrate that both curing of [URE3] and enhancement of [PSI ] in the presence of excess Cur1 are counteracted by the cochaperone Hsp40-Sis1 in a dosage-dependent manner, and show that the effect of Cur1 on prions parallels effects of the attachment of nuclear localization signal to Sis1, indicating that Cur1 acts on prions via its previously reported ability to relocalize Sis1 from the cytoplasm to nucleus. This shows that the direction in which Cur1 influences a prion depends on how this specific prion responds to relocalization of Sis1.
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http://dx.doi.org/10.1111/mmi.13697DOI Listing
July 2017

In memory of Susan Lindquist (1949-2016).

Authors:
Yury O Chernoff

Prion 2017 01;11(1):1-3

a School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA; Laboratory of Amyloid Biology and Institute of Translational Biomedicine, St. Petersburg State University , St. Petersburg , Russia.

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http://dx.doi.org/10.1080/19336896.2017.1285618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360134PMC
January 2017

Yeast Short-Lived Actin-Associated Protein Forms a Metastable Prion in Response to Thermal Stress.

Cell Rep 2017 01;18(3):751-761

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address:

Self-perpetuating ordered protein aggregates (amyloids and prions) are associated with a variety of neurodegenerative disorders. Although environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. We have employed endogenous yeast prions as a model system to study environmental control of amyloid formation. A short-lived actin-associated yeast protein Lsb2 can trigger prion formation by other proteins in a mode regulated by the cytoskeleton and ubiquitin-dependent processes. Here, we show that such a heterologous prion induction is due to the ability of Lsb2 to form a transient prion state, generated in response to thermal stress. Evolutionary acquisition of prion-inducing activity by Lsb2 is traced to a single amino acid change, coinciding with the acquisition of thermotolerance in the Saccharomyces yeast lineage. This raises the intriguing possibility that the transient prion formation could aid in functioning of Lsb2 at higher temperatures.
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http://dx.doi.org/10.1016/j.celrep.2016.12.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267347PMC
January 2017

Prions, Chaperones, and Proteostasis in Yeast.

Cold Spring Harb Perspect Biol 2017 Feb 1;9(2). Epub 2017 Feb 1.

School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332-2000.

Prions are alternatively folded, self-perpetuating protein isoforms involved in a variety of biological and pathological processes. Yeast prions are protein-based heritable elements that serve as an excellent experimental system for studying prion biology. The propagation of yeast prions is controlled by the same Hsp104/70/40 chaperone machinery that is involved in the protection of yeast cells against proteotoxic stress. Ribosome-associated chaperones, proteolytic pathways, cellular quality-control compartments, and cytoskeletal networks influence prion formation, maintenance, and toxicity. Environmental stresses lead to asymmetric prion distribution in cell divisions. Chaperones and cytoskeletal proteins mediate this effect. Overall, this is an intimate relationship with the protein quality-control machinery of the cell, which enables prions to be maintained and reproduced. The presence of many of these same mechanisms in higher eukaryotes has implications for the diagnosis and treatment of mammalian amyloid diseases.
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http://dx.doi.org/10.1101/cshperspect.a023663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5287078PMC
February 2017

Strain conformation controls the specificity of cross-species prion transmission in the yeast model.

Prion 2016 07;10(4):269-82

a Laboratory of Amyloid Biology, St. Petersburg State University , St. Petersburg , Russia.

Transmissible self-assembled fibrous cross-β polymer infectious proteins (prions) cause neurodegenerative diseases in mammals and control non-Mendelian heritable traits in yeast. Cross-species prion transmission is frequently impaired, due to sequence differences in prion-forming proteins. Recent studies of prion species barrier on the model of closely related yeast species show that colocalization of divergent proteins is not sufficient for the cross-species prion transmission, and that an identity of specific amino acid sequences and a type of prion conformational variant (strain) play a major role in the control of transmission specificity. In contrast, chemical compounds primarily influence transmission specificity via favoring certain strain conformations, while the species origin of the host cell has only a relatively minor input. Strain alterations may occur during cross-species prion conversion in some combinations. The model is discussed which suggests that different recipient proteins can acquire different spectra of prion strain conformations, which could be either compatible or incompatible with a particular donor strain.
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http://dx.doi.org/10.1080/19336896.2016.1204060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082969PMC
July 2016

Distinct types of translation termination generate substrates for ribosome-associated quality control.

Nucleic Acids Res 2016 08 20;44(14):6840-52. Epub 2016 Jun 20.

Department of Cell Biology, Rowan University, School of Osteopathic Medicine, Stratford, NJ 08084, USA.

Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45-Sup35 (eRF1-eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45-Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control.
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http://dx.doi.org/10.1093/nar/gkw566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001609PMC
August 2016

Are there prions in plants?

Authors:
Yury O Chernoff

Proc Natl Acad Sci U S A 2016 May 23;113(22):6097-9. Epub 2016 May 23.

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-2000; Laboratory of Amyloid Biology & Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg 199034, Russia

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http://dx.doi.org/10.1073/pnas.1605671113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896674PMC
May 2016

Dual role of ribosome-associated chaperones in prion formation and propagation.

Curr Genet 2016 Nov 11;62(4):677-685. Epub 2016 Mar 11.

School of Biology, Georgia Institute of Technology, 950 Atlantic Drive, Engineered Biosystems Building, M/C 2000, Atlanta, GA, 30332-2000, USA.

Chaperones of the diverse ubiquitous Hsp70 family are involved in the regulation of ordered self-perpetuating protein aggregates (amyloids and prions), implicated in both devastating diseases and protein-based inheritance. Yeast ribosome-associated chaperone complex (RAC), composed of the Hsp40 protein Zuo1 and non-canonical Hsp70 protein Ssz1, mediates association of the Hsp70 chaperone Ssb with translating ribosomes. Ssb participates in co-translational protein folding, regulation of premature translation termination, and ribosome biogenesis. The loss of Ssb or disruption of RAC results in the increased formation of [PSI ], a prion form of the translation termination factor Sup35 (eRF3). This implicates co-translational protein misfolding in de novo prion formation. However, RAC disruption also destabilizes pre-existing [PSI ] prions, as Ssb, released from ribosomes to the cytosol in the absence of RAC, antagonizes the function of the major cytosolic chaperone, Ssa, in prion propagation. The mechanism of the Ssa/Ssb antagonism is currently under investigation and may include a competition for substrates and/or co-chaperones. Notably, yeast cells with wild-type RAC also release Ssb to the cytosol in certain unfavorable growth conditions, and Ssb contributes to increased prion loss in these conditions. This indicates that the circulation of Ssb between the ribosome and cytosol may serve as a physiological regulator of the formation and propagation of self-perpetuating protein aggregates. Indeed, RAC and Ssb modulate toxicity of some aggregating proteins in yeast. Mammalian cells lack the Ssb ortholog but contain a RAC counterpart, apparently recruiting other Hsp70 protein(s). Thus, amyloid modulation by ribosome-associated chaperones could be applicable beyond yeast.
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http://dx.doi.org/10.1007/s00294-016-0586-2DOI Listing
November 2016

The call of the unknown: The story of [PSI(+)].

Authors:
Yury O Chernoff

Prion 2015 ;9(5):315-7

a School of Biology; Georgia Institute of Technology ; Atlanta , GA USA ;

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http://dx.doi.org/10.1080/19336896.2015.1112656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4964858PMC
September 2016

Contributions of the Prion Protein Sequence, Strain, and Environment to the Species Barrier.

J Biol Chem 2016 Jan 12;291(3):1277-88. Epub 2015 Nov 12.

Biology, Georgia Institute of Technology, Atlanta, Georgia 30332 and the Laboratory of Amyloid Biology and Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg 199034, Russia

Amyloid propagation requires high levels of sequence specificity so that only molecules with very high sequence identity can form cross-β-sheet structures of sufficient stringency for incorporation into the amyloid fibril. This sequence specificity presents a barrier to the transmission of prions between two species with divergent sequences, termed a species barrier. Here we study the relative effects of protein sequence, seed conformation, and environment on the species barrier strength and specificity for the yeast prion protein Sup35p from three closely related species of the Saccharomyces sensu stricto group; namely, Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces paradoxus. Through in vivo plasmid shuffle experiments, we show that the major characteristics of the transmission barrier and conformational fidelity are determined by the protein sequence rather than by the cellular environment. In vitro data confirm that the kinetics and structural preferences of aggregation of the S. paradoxus and S. bayanus proteins are influenced by anions in accordance with their positions in the Hofmeister series, as observed previously for S. cerevisiae. However, the specificity of the species barrier is primarily affected by the sequence and the type of anion present during the formation of the initial seed, whereas anions present during the seeded aggregation process typically influence kinetics rather than the specificity of prion conversion. Therefore, our work shows that the protein sequence and the conformation variant (strain) of the prion seed are the primary determinants of cross-species prion specificity both in vivo and in vitro.
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http://dx.doi.org/10.1074/jbc.M115.684100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714215PMC
January 2016

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils.

EMBO J 2015 Sep 24;34(18):2363-82. Epub 2015 Aug 24.

Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.
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http://dx.doi.org/10.15252/embj.201591245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570522PMC
September 2015

Feedback control of prion formation and propagation by the ribosome-associated chaperone complex.

Mol Microbiol 2015 May 11;96(3):621-32. Epub 2015 Mar 11.

School of Biology and Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA; Laboratory of Amyloid Biology, St. Petersburg State University, St. Petersburg, Russia.

Cross-beta fibrous protein aggregates (amyloids and amyloid-based prions) are found in mammals (including humans) and fungi (including yeast), and are associated with both diseases and heritable traits. The Hsp104/70/40 chaperone machinery controls propagation of yeast prions. The Hsp70 chaperones Ssa and Ssb show opposite effects on [PSI(+)], a prion form of the translation termination factor Sup35 (eRF3). Ssb is bound to translating ribosomes via ribosome-associated complex (RAC), composed of Hsp40-Zuo1 and Hsp70-Ssz1. Here we demonstrate that RAC disruption increases de novo prion formation in a manner similar to Ssb depletion, but interferes with prion propagation in a manner similar to Ssb overproduction. Release of Ssb into the cytosol in RAC-deficient cells antagonizes binding of Ssa to amyloids. Thus, propagation of an amyloid formed because of lack of ribosome-associated Ssb can be counteracted by cytosolic Ssb, generating a feedback regulatory circuit. Release of Ssb from ribosomes is also observed in wild-type cells during growth in poor synthetic medium. Ssb is, in a significant part, responsible for the prion destabilization in these conditions, underlining the physiological relevance of the Ssb-based regulatory circuit.
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http://dx.doi.org/10.1111/mmi.12960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452204PMC
May 2015