DNAJB chaperones suppress destabilised protein aggregation via a region unique from that used to inhibit amyloidogenesis

Disturbances to protein homeostasis (proteostasis) can lead to protein aggregation and inclusion formation, processes associated with a variety of neurodegenerative disorders. DNAJBs are molecular chaperones which have been identified as potent suppressors of disease-related protein aggregation. In this work, a destabilised isoform of firefly luciferase (R188Q/R261Q Fluc; FlucDM) was overexpressed in cells to assess the capacity of DNAJBs to inhibit inclusion formation. Co-expression of all DNAJBs tested significantly inhibited the intracellular aggregation of FlucDM. Moreover, we show that DNAJBs suppress aggregation by supporting the Hsp70-dependent degradation of FlucDM via the proteasome. The serine-rich stretch in DNAJB6 and DNAJB8, essential for preventing fibrillar aggregation, is not involved in the suppression of FlucDM inclusion formation. Conversely, deletion of the C-terminal TTK-LKS motif in DNAJB6 and DNAJB8, a region not required to suppress polyQ aggregation, abolished its ability to inhibit inclusion formation by FlucDM. Thus, our data suggest that DNAJB6 and DNAJB8 possess two distinct regions for binding substrates, one that is responsible for binding β-hairpins that form during amyloid formation and another that interacts with exposed hydrophobic patches in aggregation-prone clients.

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DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation

10.1101/2020.10.08.326280 ◽

2020 ◽

Author(s):

Shannon McMahon ◽

Steven Bergink ◽

Harm H. Kampinga ◽

Heath Ecroyd

Keyword(s):

Protein Aggregation ◽

Molecular Chaperones ◽

Firefly Luciferase ◽

Amyloid Formation ◽

Protein Homeostasis ◽

Formation Processes ◽

Inclusion Formation ◽

Different Types ◽

Summary Statement ◽

Client Proteins

AbstractDisturbances to protein homeostasis (proteostasis) can lead to protein aggregation and inclusion formation, processes associated with a variety of neurodegenerative disorders. DNAJBs are molecular chaperones previously identified as potent suppressors of disease-related protein aggregation. In this work, we over-expressed a destabilised isoform of firefly luciferase (R188Q/R261Q Fluc; FlucDM) in cells to assess the capacity of DNAJBs to inhibit inclusion formation. Co-expression of all DNAJBs tested significantly inhibited the intracellular aggregation of FlucDM. Moreover, we show that DNAJBs suppress aggregation by supporting the Hsp70-dependent degradation of FlucDM via the proteasome. The serine-rich stretch in DNAJB6 and DNAJB8, essential for preventing fibrillar aggregation, is not involved in the suppression of FlucDM inclusion formation. Conversely, deletion of the C-terminal TTK-LKS region in DNAJB8, a region not required to suppress polyQ aggregation, abolished its ability to inhibit inclusion formation by FlucDM. Thus, our data suggest that DNAJB6 and DNAJB8 possess two distinct domains involved in the inhibition of protein aggregation, one responsible for binding to β-hairpins that form during amyloid formation and another that mediates the degradation of destabilised client proteins via the proteasome.Summary statementSpecialised DNAJB molecular chaperones are potent suppressors of protein aggregation and interact with different types of client proteins via distinct C-terminal regions

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Suppression of aggregate and amyloid formation by a novel intrinsically disordered region in metazoan Hsp110 chaperones

10.1101/2021.01.13.426581 ◽

2021 ◽

Author(s):

Unekwu M. Yakubu ◽

Kevin A. Morano

Keyword(s):

Protein Aggregation ◽

Molecular Chaperones ◽

Citrate Synthase ◽

Neuronal Cell ◽

Amyloid Formation ◽

Nucleotide Exchange ◽

Aggregation Assay ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region

AbstractMolecular chaperones maintain protein homeostasis (proteostasis) by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to the onset of numerous neurodegenerative disorders including Alzheimer’s, Parkinson’s, and Huntington’s disease. Hsp110 is related to the canonical Hsp70 class of protein folding molecular chaperones and interacts with Hsp70 as a nucleotide exchange factor (NEF), promoting rapid cycling of ADP for ATP. In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate binding domain (SBD) whose biological roles remain undefined. Previous work in Drosophila melanogaster has shown that loss of the sole Hsp110 gene (Hsc70cb) accelerates the aggregation of polyglutamine (polyQ)-expanded human Huntingtin, while its overexpression protects against polyQ-mediated neuronal cell death. We hypothesize that in addition to its role as an Hsp70 NEF, Drosophila Hsp110 may function in the fly as a protective protein “holdase”, preventing the aggregation of unfolded polypeptides via the SBD-β subdomain. Using an in vitro protein aggregation assay we demonstrate for the first time that Drosophila Hsp110 effectively prevents aggregation of the model substrate citrate synthase. We also report the discovery of a redundant and heretofore unknown potent holdase capacity in a 138 amino-acid region of Hsp110 carboxyl-terminal to both SBD-β and SBD-α (henceforth called the C-terminal extension). This sequence is highly conserved in metazoan Hsp110 genes, completely absent from fungal representatives, including Saccharomyces cerevisiae SSE1, and is computationally predicted to contain an intrinsically disordered region (IDR). We demonstrate that this IDR sequence within the human Hsp110s, Apg-1 and Hsp105α, inhibits the formation of amyloid Aβ-42 and α-synuclein fibrils in vitro but cannot mediate fibril disassembly. Together these findings demonstrate the existence of a second independent, passive holdase property of metazoan Hsp110 chaperones capable of suppressing both general protein aggregation and amyloidogenesis and raise the possibility of exploitation of this IDR for therapeutic benefit in combating neurodegenerative disease.

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The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation

10.1101/2020.12.09.417709 ◽

2020 ◽

Author(s):

Anita Pras ◽

Bert Houben ◽

Francesco A. Aprile ◽

Renée Seinstra ◽

Rodrigo Gallardo ◽

...

Keyword(s):

Protein Aggregation ◽

Structural Changes ◽

Colloidal Stability ◽

Positive Charge ◽

Amyloid Formation ◽

Related Protein ◽

Age Related ◽

Related Proteins ◽

Protein Toxicity ◽

Charge Interactions

AbstractWhile aggregation-prone proteins are known to accelerate ageing and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A and SERF2 have recently been identified as cellular modifiers of such cytotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with specific patterns of negatively charged and hydrophobic, aromatic amino acids. The absence of such patterns, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In a protein aggregation model in the nematode C. elegans, protein aggregation was suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that charge interactions are required for MOAG-4 and SERF2 to promote aggregation. Such charged interactions might accelerate the primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our finding that negatively charged segments are overrepresented in amyloid-forming proteins suggests that inhibition of charge interactions deserves exploration as a strategy to target age-related protein toxicity.Significance StatementHow aging causes relatively common diseases such as Alzheimer’s and Parkinson’s is still a mystery. Since toxic structural changes in proteins are likely to be responsible, we investigated biological mechanisms that could drive such changes. We made use of a modifying factor called SERF2, which accelerates structural changes and aggregation of several disease-related proteins. Through a peptide-binding screen, we found that SERF2 acts on negatively charged protein regions. The abundance of such regions in the disease-related proteins explains why SERF has its effect. Removing positive charge in SERF was sufficient to suppress protein aggregation in models for disease. We propose that blocking charge-interactions with SERF or other modifiers could serve as a general approach to treat age-related protein toxicity.

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Pathways of cellular proteostasis in aging and disease

The Journal of Cell Biology ◽

10.1083/jcb.201709072 ◽

2017 ◽

Vol 217 (1) ◽

pp. 51-63 ◽

Cited By ~ 246

Author(s):

Courtney L. Klaips ◽

Gopal Gunanathan Jayaraj ◽

F. Ulrich Hartl

Keyword(s):

Protein Synthesis ◽

Molecular Chaperones ◽

Neurodegenerative Disorders ◽

Cellular Protein ◽

Protein Homeostasis ◽

Precise Control ◽

Parkinson’S Diseases ◽

Age Dependent ◽

Network Coordinates ◽

Dependent Failure

Ensuring cellular protein homeostasis, or proteostasis, requires precise control of protein synthesis, folding, conformational maintenance, and degradation. A complex and adaptive proteostasis network coordinates these processes with molecular chaperones of different classes and their regulators functioning as major players. This network serves to ensure that cells have the proteins they need while minimizing misfolding or aggregation events that are hallmarks of age-associated proteinopathies, including neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. It is now clear that the capacity of cells to maintain proteostasis undergoes a decline during aging, rendering the organism susceptible to these pathologies. Here we discuss the major proteostasis pathways in light of recent research suggesting that their age-dependent failure can both contribute to and result from disease. We consider different strategies to modulate proteostasis capacity, which may help develop urgently needed therapies for neurodegeneration and other age-dependent pathologies.

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Hsp40s display class-specific binding profiles, serving complementary roles in the prevention of tau amyloid formation

10.1101/2021.04.11.439324 ◽

2021 ◽

Author(s):

Rose Irwin ◽

Ofrah Faust ◽

Ivana Petrovic ◽

Sharion Grayer Wolf ◽

Hagen Hofmann ◽

...

Keyword(s):

Molecular Chaperones ◽

Specific Binding ◽

Small Heat Shock Protein ◽

Fiber Formation ◽

Amyloid Formation ◽

Protein Homeostasis ◽

Protein Tau ◽

Amyloid Fiber Formation ◽

Tau Aggregation ◽

Insight Into

The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles, forming insoluble, amyloid-type aggregates associated with neurodegenerative conditions, such as Alzheimer's disease. Tau aggregation, however, can be prevented in the cell by a class of proteins known as molecular chaperones, which play important roles in maintaining protein homeostasis. While numerous chaperones are known to interact with tau, though, little is known about the detailed mechanisms by which these prevent tau aggregation. Here, we describe the effects of the ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid fiber formation and compare these to the well-studied small heat shock protein HSPB1. We find that each chaperone prevents tau aggregation differently, by interacting with distinct sets of tau species along the aggregation pathway and thereby affecting their incorporation into fibers. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone tau conformers and even mature fibers, thus efficiently preventing formation of tau amyloids. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, different site in CTDI. These results provide important insight into the molecular mechanism by which the different members of the Hsp40 chaperone family counteract the formation, propagation, and toxicity of tau aggregates. Furthermore, our findings highlight the fact that chaperones from different families and different classes play distinct, but complementary roles in preventing pathological protein aggregation.

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The physical chemistry of the amyloid phenomenon: thermodynamics and kinetics of filamentous protein aggregation

Essays in Biochemistry ◽

10.1042/bse0560011 ◽

2014 ◽

Vol 56 ◽

pp. 11-39 ◽

Cited By ~ 31

Author(s):

Alexander K. Buell ◽

Christopher M. Dobson ◽

Tuomas P.J. Knowles

Keyword(s):

Protein Aggregation ◽

Molecular Chaperones ◽

Amyloid Fibrils ◽

Theoretical Modelling ◽

Amyloid Formation ◽

Recent Developments ◽

The Individual ◽

Kinetics Of ◽

Soluble State ◽

Insight Into

In this chapter, we present an overview of the kinetics and thermodynamics of protein aggregation into amyloid fibrils. The perspective we adopt is largely experimental, but we also discuss recent developments in data analysis and we show that only a combination of well-designed experiments with appropriate theoretical modelling is able to provide detailed mechanistic insight into the complex pathways of amyloid formation. In the first part of the chapter, we describe measurements of the thermodynamic stability of the amyloid state with respect to the soluble state of proteins, as well as the magnitude and origin of this stability. In the second part, we discuss in detail the kinetics of the individual molecular steps in the overall mechanism of the conversion of soluble protein into amyloid fibrils. Finally, we highlight the effects of external factors, such as salt type and concentration, chemical denaturants and molecular chaperones on the kinetics of aggregation.

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Deubiquitylating enzymes in neuronal health and disease

Cell Death and Disease ◽

10.1038/s41419-020-03361-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Fatima Amer-Sarsour ◽

Alina Kordonsky ◽

Yevgeny Berdichevsky ◽

Gali Prag ◽

Avraham Ashkenazi

Keyword(s):

Human Brain ◽

Neurodegenerative Disorders ◽

Structure And Function ◽

Pivotal Role ◽

Protein Homeostasis ◽

Neuronal Function ◽

Health And Disease ◽

And Function

AbstractUbiquitylation and deubiquitylation play a pivotal role in protein homeostasis (proteostasis). Proteostasis shapes the proteome landscape in the human brain and its impairment is linked to neurodevelopmental and neurodegenerative disorders. Here we discuss the emerging roles of deubiquitylating enzymes in neuronal function and survival. We provide an updated perspective on the genetics, physiology, structure, and function of deubiquitylases in neuronal health and disease.

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Valorization of Apple Peels through the Study of the Effects on the Amyloid Aggregation Process of κ-Casein

Molecules ◽

10.3390/molecules26082371 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2371

Author(s):

Valeria Guarrasi ◽

Giacoma Cinzia Rappa ◽

Maria Assunta Costa ◽

Fabio Librizzi ◽

Marco Raimondo ◽

...

Keyword(s):

Protein Aggregation ◽

Environmental Sustainability ◽

Bovine Milk ◽

Amyloid Formation ◽

Aggregation Process ◽

Amyloid Aggregation ◽

Social Challenges ◽

Force Microscopy ◽

Atomic Force ◽

Apple Peels

Waste valorization represents one of the main social challenges when promoting a circular economy and environmental sustainability. Here, we evaluated the effect of the polyphenols extracted from apple peels, normally disposed of as waste, on the amyloid aggregation process of κ-casein from bovine milk, a well-used amyloidogenic model system. The effect of the apple peel extract on protein aggregation was examined using a thioflavin T fluorescence assay, Congo red binding assay, circular dichroism, light scattering, and atomic force microscopy. We found that the phenolic extract from the peel of apples of the cultivar “Fuji”, cultivated in Sicily (Caltavuturo, Italy), inhibited κ-casein fibril formation in a dose-dependent way. In particular, we found that the extract significantly reduced the protein aggregation rate and inhibited the secondary structure reorganization that accompanies κ-casein amyloid formation. Protein-aggregated species resulting from the incubation of κ-casein in the presence of polyphenols under amyloid aggregation conditions were reduced in number and different in morphology.

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