scholarly journals Identification of subfunctionalized aggregate-remodeling J-domain proteins in plants

2020 ◽  
Author(s):  
Yogesh Tak ◽  
Silviya S. Lal ◽  
Shilpa Gopan ◽  
Madhumitha Balakrishnan ◽  
Amit K. Verma ◽  
...  
Keyword(s):  
Protein Quality ◽  
Expression Patterns ◽  
Protein Aggregates ◽  
Cellular Protein ◽  
Multiple Model ◽  
Protein Aggregate ◽  
Cytotoxic Protein ◽  
J Domain ◽  
Critical Components

AbstractHsp70s and J-domain proteins (JDPs) are among the most critical components of the cellular protein quality control machinery, playing crucial roles in preventing and solubilizing cytotoxic protein aggregates. Bacteria, yeast and plants additionally have large, multimeric Hsp100-class disaggregases which, allow the resolubilization of otherwise “dead-end” aggregates, including amyloids. JDPs interact with aggregated proteins and specify the aggregate remodeling activities of Hsp70s and Hsp100s. Plants have a complex network of cytosolic Hsp70s and JDPs, however the aggregate remodeling properties of plant JDPs are not well understood. Here we identify evolutionary-conserved Class II JDPs in the model plant Arabidopsis thaliana with distinct aggregate remodeling functionalities. We identify eight plant orthologs of the yeast protein, Sis1, the principal JDP responsible for directing the yeast chaperone machinery for remodeling protein aggregates. Expression patterns vary dramatically among the eight paralogous proteins under a variety of stress conditions, indicating their subfunctionalization to address distinct stressors. Consistent with a role in solubilizing cytotoxic protein aggregates, six of these plant JDPs associate with heat-induced protein aggregates in vivo as well as colocalize with plant Hsp101 to distinct heat-induced protein aggregate centers. Finally, we show that these six JDPs can differentially remodel multiple model protein aggregates in yeast confirming their involvement in aggregate resolubilization. These results demonstrate that compared to complex metazoans, plants have a robust network of JDPs involved in aggregate remodeling activities with the capacity to process a variety of protein aggregate conformers.

scholarly journals Protein Aggregation is Associated with Acinetobacter baumannii Desiccation Tolerance

2020 ◽  
Vol 8 (3) ◽  
pp. 343 ◽  
Author(s):  
Xun Wang ◽  
Cody G. Cole ◽  
Cory D. DuPai ◽  
Bryan W. Davies
Keyword(s):  
Acinetobacter Baumannii ◽  
Desiccation Tolerance ◽  
Bacterial Pathogen ◽  
Protein Aggregates ◽  
Cellular Protein ◽  
Aggregate Formation ◽  
Growth Phases ◽  
Protein Aggregate ◽  
Factors Influencing ◽  
Cellular Aggregates

Desiccation tolerance has been implicated as an important characteristic that potentiates the spread of the bacterial pathogen Acinetobacter baumannii on dry surfaces. Here we explore several factors influencing desiccation survival of A. baumannii. At the macroscale level, we find that desiccation tolerance is influenced by cell density and growth phase. A transcriptome analysis indicates that desiccation represents a unique state for A. baumannii compared to commonly studied growth phases and strongly influences pathways responsible for proteostasis. Remarkably, we find that an increase in total cellular protein aggregates, which is often considered deleterious, correlates positively with the ability of A. baumannii to survive desiccation. We show that inducing protein aggregate formation prior to desiccation increases survival and, importantly, that proteins incorporated into cellular aggregates can retain activity. Our results suggest that protein aggregates may promote desiccation tolerance in A. baumannii through preserving and protecting proteins from damage during desiccation until rehydration occurs.

scholarly journals Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-based Disaggregase

2021 ◽  
Vol 22 (23) ◽  
pp. 12983
Author(s):  
Aitor Franco ◽  
Jorge Cuéllar ◽  
José Ángel Fernández-Higuero ◽  
Igor de la Arada ◽  
Natalia Orozco ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Quality ◽  
Mechanical Action ◽  
Cellular Protein ◽  
Type B ◽  
Post Translational Modifications ◽  
Lateral Association ◽  
N Terminus ◽  
Protein Quality Control System

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.

scholarly journals Amyotrophic Lateral Sclerosis and Autophagy: Dysfunction and Therapeutic Targeting

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2413
Author(s):  
Azin Amin ◽  
Nirma D. Perera ◽  
Philip M. Beart ◽  
Bradley J. Turner ◽  
Fazel Shabanpoor
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Protein Aggregates ◽  
Misfolded Proteins ◽  
Protein Aggregate ◽  
In Vivo Models ◽  
Primary Stress ◽  
Lateral Sclerosis

Over the past 20 years, there has been a drastically increased understanding of the genetic basis of Amyotrophic Lateral Sclerosis. Despite the identification of more than 40 different ALS-causing mutations, the accumulation of neurotoxic misfolded proteins, inclusions, and aggregates within motor neurons is the main pathological hallmark in all cases of ALS. These protein aggregates are proposed to disrupt cellular processes and ultimately result in neurodegeneration. One of the main reasons implicated in the accumulation of protein aggregates may be defective autophagy, a highly conserved intracellular “clearance” system delivering misfolded proteins, aggregates, and damaged organelles to lysosomes for degradation. Autophagy is one of the primary stress response mechanisms activated in highly sensitive and specialised neurons following insult to ensure their survival. The upregulation of autophagy through pharmacological autophagy-inducing agents has largely been shown to reduce intracellular protein aggregate levels and disease phenotypes in different in vitro and in vivo models of neurodegenerative diseases. In this review, we explore the intriguing interface between ALS and autophagy, provide a most comprehensive summary of autophagy-targeted drugs that have been examined or are being developed as potential treatments for ALS to date, and discuss potential therapeutic strategies for targeting autophagy in ALS.

scholarly journals Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells inCaulobacter crescentus

2018 ◽  
Author(s):  
Frederic D. Schramm ◽  
Kristen Schroeder ◽  
Jonatan Alvelid ◽  
Ilaria Testa ◽  
Kristina Jonas
Keyword(s):  
Protein Quality ◽  
Caulobacter Crescentus ◽  
Protein Aggregates ◽  
Time Lapse ◽  
Daughter Cells ◽  
Chaperone Dnak ◽  
Kinetics Of ◽  
Successive Division ◽  
Moderate Stress

AbstractAll living cells must deal with protein aggregation, which can occur as a result of experiencing stress. In the bacteriaEscherichia coliandMycobacterium smegmatis, aggregates collect at the cell poles and are retained over consecutive cell divisions only in the daughter cell that inherits the old pole, resulting in aggregation-free progeny within a few generations. Here we have studied thein vivokinetics of aggregate formation and clearance following heat and antibiotic stress inCaulobacter crescentus, which divides by a pre-programmed asymmetric cell cycle. Unexpectedly, we find that aggregates do not preferentially collect at the cell poles, but form as multiple distributed foci throughout the cell volume. Time-lapse microscopy revealed that under moderate stress, the majority of protein aggregates are short-lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality machinery results in long-lived protein aggregates, which individual cells can only clear by passing on to their progeny. Importantly, these persistent aggregates are neither collected at the old pole over multiple generations nor inherited exclusively by the old pole-inheriting stalked cell, but instead are partitioned between both daughter cells during successive division events in the same ratio. Our data indicate that this symmetric mode of aggregate inheritance is driven by the elongation and division of the growing mother cell. In conclusion, our study revealed a new pattern of aggregate inheritance in bacteria.

scholarly journals Proteomic Analysis of Mitochondrial Protein Turnover: Identification of Novel Substrate Proteins of the Matrix Protease Pim1

2006 ◽  
Vol 26 (3) ◽  
pp. 762-776 ◽  
Author(s):  
Tamara Major ◽  
Birgit von Janowsky ◽  
Thomas Ruppert ◽  
Axel Mogk ◽  
Wolfgang Voos
Keyword(s):  
Protein Turnover ◽  
Protein Quality ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Differential Susceptibility ◽  
Cellular Protein ◽  
Major Influence ◽  
Mitochondrial Functions ◽  
Substrate Proteins

ABSTRACT ATP-dependent oligomeric proteases are major components of cellular protein quality control systems. To investigate the role of proteolytic processes in the maintenance of mitochondrial functions, we analyzed the dynamic behavior of the mitochondrial proteome of Saccharomyces cerevisiae by two-dimensional (2D) polyacrylamide gel electrophoresis. By a characterization of the influence of temperature on protein turnover in isolated mitochondria, we were able to define four groups of proteins showing a differential susceptibility to proteolysis. The protein Pim1/LON has been shown to be the main protease in the mitochondrial matrix responsible for the removal of damaged or nonnative proteins. To assess the substrate range of Pim1 under in vivo conditions, we performed a quantitative comparison of the 2D protein spot patterns between wild-type and pim1Δ mitochondria. We were able to identify a novel subset of mitochondrial proteins that are putative endogenous substrates of Pim1. Using an in organello degradation assay, we confirmed the Pim1-specific, ATP-dependent proteolysis of the newly identified substrate proteins. We could demonstrate that the functional integrity of the Pim1 substrate proteins, in particular, the presence of intact prosthetic groups, had a major influence on the susceptibility to proteolysis.

scholarly journals Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

2011 ◽  
Vol 22 (5) ◽  
pp. 541-554 ◽  
Author(s):  
Tom Bender ◽  
Ilka Lewrenz ◽  
Sebastian Franken ◽  
Catherina Baitzel ◽  
Wolfgang Voos
Keyword(s):  
Quality Control ◽  
Protein Aggregation ◽  
Elevated Temperatures ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Cellular Protein ◽  
Control Measures ◽  
Mitochondrial Enzymes ◽  
Induced Aggregation

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

scholarly journals Novel proteostasis reporter mouse reveals different effects of cytoplasmic and nuclear aggregates on protein quality control in neurons

2020 ◽  
Author(s):  
Sonja Blumenstock ◽  
Elena Katharina Schulz-Trieglaff ◽  
Anna-Lena Bolender ◽  
Kerstin Voelkl ◽  
Paul Lapios ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Cellular Protein ◽  
Aging Brain ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Age Related ◽  
Reporter Mice

AbstractThe cellular protein quality control machinery is important for preventing protein misfolding and aggregation, and decline in protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how proteostasis capacity of neurons changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc), a conformationally unstable protein that requires chaperones for proper folding and sensitively reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked impairment in proteostasis in tauopathy mice, but not in Huntington’s disease mice. Mechanistic investigations in primary neuronal cultures demonstrate that cytoplasmic, but not nuclear, aggregates cause defects of cellular protein quality control. Thus, the Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.

scholarly journals Domain Organization of the UBX Domain Containing Protein 9 and Analysis of Its Interactions With the Homohexameric AAA + ATPase p97 (Valosin-Containing Protein)

2021 ◽  
Vol 9 ◽  
Author(s):  
Jana Riehl ◽  
Ramesh Rijal ◽  
Leonie Nitz ◽  
Christoph S. Clemen ◽  
Andreas Hofmann ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Model ◽  
Protein Quality ◽  
Quaternary Structure ◽  
Cellular Protein ◽  
Protein Family ◽  
Actin Binding ◽  
Native Page ◽  
Aaa Atpase

The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384–466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β0-α–1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261–573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261–573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1’ intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.

Abstract 393: The Intercalated Disc Protein Myozap: A Novel Player in Cardiac Proteinopathy

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Ashraf Y Rangrez ◽  
Derk Frank ◽  
Liam Cassidy ◽  
Lynn Christen ◽  
Inka Geurink ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Protein Aggregation ◽  
Protein Quality ◽  
Transcriptome Profiling ◽  
Protein Aggregates ◽  
Intercalated Disc ◽  
Protein Aggregate ◽  
General Role ◽  
Id Protein

Background: A growing number of cardiac muscle diseases are characterized by depositions of misfolded proteins, including cardiac amyloidosis and desmin-releated cardiomyopathy (DRM). The continued presence and chronic accumulation of misfolded or unfolded proteins can lead to aggregation and/or the formation of soluble peptides that are proteotoxic. This in turn leads to compromised protein quality control and precipitates a downward spiral of the cell’s ability to maintain homeostasis and may eventually result in cell death. We recently identified massive protein aggregates in the hearts of transgenic mice overexpressing the intercalated disc (ID) protein myozap (Myozap-tg). We now sought to investigate the precise composition of these aggregates and the role of Myozap in other proteinopathies such as DRM. Methods and Results: We employed multi-dimensional proteomics, transcriptomics, confocal microscopy, and molecular biology approaches to decipher the underlying causes and consequences of protein aggregate formation in Myozap-tg mice. Transcriptome profiling of these mice revealed striking upregulation of autophagy, protein synthesis, and pro-inflammatory pathways, whereas protein degradation pathways were down-regulated. Surprisingly, proteomics analyses revealed Desmin and α-crystallin B (CryAB) as the major constituents of the aggregates, which was further validated by confocal microscopy. Moreover, we identified the presence of toxic preamyloid oligomers in Myozap-tg mouse hearts, a hallmark in many protein aggregation-based diseases including DRM. Most interestingly, we also observed co-localization of Myozap with protein aggregates observed in both transgenic mouse hearts overexpressing mutant Desmin (D7) and mutant CryAB (R120G), as well as in human DRM patients. Conclusion: The present study implies a new role for Myozap, which was previously reported to affect cardiac SRF signaling: (1) Myozap accumulates in various forms of experimental and human protein aggregation cardiomyopathy, suggesting involvement in protein homoestasis. (2) The fact that Myozap is now the third ID protein (after desmin and CryAB) to cause cardiac proteinopathy points to a general role of the ID in its molecular pathogenesis.

scholarly journals Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases

2015 ◽  
Vol 112 (7) ◽  
pp. E625-E632 ◽  
Author(s):  
Itamar Cohen ◽  
Reuven Wiener ◽  
Yuval Reiss ◽  
Tommer Ravid
Keyword(s):  
Transition State ◽  
Protein Quality ◽  
Noncovalent Interactions ◽  
Cellular Protein ◽  
Ring Domain ◽  
E3 Ligases ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed “ER-associated degradation” (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed “Der3”) and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired the in vivo degradation of Doa10 substrates but had no apparent effect on the degradation of Hrd1 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the α2 helix of Ubc7 and Ub and that this transition state may be stabilized further by some E3 ligases, such as Hrd1, through additional interactions outside the RING domain.

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