Hsp70 machinery vs protein aggregation: the role of chaperones in cellular protein homeostasis

Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.

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α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast

Cells ◽

10.3390/cells10092229 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2229

Author(s):

Blagovesta Popova ◽

Dajana Galka ◽

Nicola Häffner ◽

Dan Wang ◽

Kerstin Schmitt ◽

...

Keyword(s):

Dopaminergic Neurons ◽

Cellular Protein ◽

Protein Homeostasis ◽

Wild Type ◽

Yeast Growth ◽

Independent Manner ◽

Proteasome Subunits ◽

Dependent Protein ◽

The Brain

Parkinson’s disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.

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Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling

10.1101/2021.11.30.470448 ◽

2021 ◽

Author(s):

Ziqi Lyu ◽

Melody M. Sycks ◽

Mateo F. Espinoza ◽

Khanh K. Nguyen ◽

Cheska M. Galapate ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Protein Import ◽

Living Cells ◽

Cellular Protein ◽

Protein Homeostasis ◽

Eukaryotic Proteins ◽

Dependent Protein ◽

Stress Dependent

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting, and in turn threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), are difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we demonstrate that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

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Mass spectrometry study of a transferrin-based protein drug reveals the key role of protein aggregation for successful oral delivery

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1206924109 ◽

2012 ◽

Vol 109 (34) ◽

pp. 13544-13548 ◽

Cited By ~ 13

Author(s):

C. E. Bobst ◽

S. Wang ◽

W.-C. Shen ◽

I. A. Kaltashov

Keyword(s):

Mass Spectrometry ◽

Protein Aggregation ◽

Oral Delivery ◽

Protein Drug ◽

Mass Spectrometry Study

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Mechanisms and Consequences of Protein Aggregation: The Role of Folding Intermediates

Current Protein and Peptide Science ◽

10.2174/138920309789351976 ◽

2009 ◽

Vol 10 (5) ◽

pp. 456-463 ◽

Cited By ~ 9

Author(s):

Sangita Seshadri ◽

Keith Oberg ◽

Vladimir Uversky

Keyword(s):

Protein Aggregation ◽

Folding Intermediates

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Akt2 Regulates Cardiac Metabolism and Cardiomyocyte Survival

Journal of Biological Chemistry ◽

10.1074/jbc.m513087200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32841-32851 ◽

Cited By ~ 110

Author(s):

Brian DeBosch ◽

Nandakumar Sambandam ◽

Carla Weinheimer ◽

Michael Courtois ◽

Anthony J. Muslin

Keyword(s):

Pressure Overload ◽

Cardiac Metabolism ◽

Cellular Protein ◽

Growth Responses ◽

Infarct Zone ◽

Targeted Disruption ◽

Cellular Processes ◽

Ligand Stimulation ◽

And Function

The Akt family of serine-threonine kinases participates in diverse cellular processes, including the promotion of cell survival, glucose metabolism, and cellular protein synthesis. All three known Akt family members, Akt1, Akt2 and Akt3, are expressed in the myocardium, although Akt1 and Akt2 are most abundant. Previous studies demonstrated that Akt1 and Akt3 overexpression results in enhanced myocardial size and function. Yet, little is known about the role of Akt2 in modulating cardiac metabolism, survival, and growth. Here, we utilize murine models with targeted disruption of the akt2 or the akt1 genes to demonstrate that Akt2, but not Akt1, is required for insulin-stimulated 2-[3H]deoxyglucose uptake and metabolism. In contrast, akt2-/- mice displayed normal cardiac growth responses to provocative stimulation, including ligand stimulation of cultured cardiomyocytes, pressure overload by transverse aortic constriction, and myocardial infarction. However, akt2-/- mice were found to be sensitized to cardiomyocyte apoptosis in response to ischemic injury, and apoptosis was significantly increased in the peri-infarct zone of akt2-/- hearts 7 days after occlusion of the left coronary artery. These results implicate Akt2 in the regulation of cardiomyocyte metabolism and survival.

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