scholarly journals Genome-wide RNAi screen and in vivo protein aggregation reporters identify degradation of damaged proteins as an essential hypertonic stress response

2008 ◽  
Vol 295 (6) ◽  
pp. C1488-C1498 ◽  
Author(s):  
Keith P. Choe ◽  
Kevin Strange
Keyword(s):  
Protein Aggregation ◽  
Ubiquitin Proteasome System ◽  
Protein Damage ◽  
Cell Shrinkage ◽  
Protein Aggregate ◽  
Fluorescent Reporter ◽  
Hypertonic Stress ◽  
C Elegans ◽  
Endogenous Proteins

The damaging effects of hypertonic stress on cellular proteins are poorly defined, and almost nothing is known about the pathways that detect and repair hypertonicity-induced protein damage. To begin addressing these problems, we screened ∼19,000 Caenorhabditis elegans genes by RNA interference (RNAi) feeding and identified 40 that are essential for survival during acute hypertonic stress. Half (20 of 40) of these genes encode proteins that function to detect, transport, and degrade damaged proteins, including components of the ubiquitin-proteasome system, endosomal sorting complexes, and lysosomes. High-molecular-weight ubiquitin conjugates increase during hypertonic stress, suggesting a global change in the ubiquitinylation state of endogenous proteins. Using a polyglutamine-containing fluorescent reporter, we demonstrate that cell shrinkage induces rapid protein aggregation in vivo and that many of the genes that are essential for survival during hypertonic stress function to prevent accumulation of aggregated proteins. High levels of urea, a strong protein denaturant, do not cause aggregation, suggesting that factors such as macromolecular crowding also contribute to protein aggregate formation during cell shrinkage. Acclimation of C. elegans to mild hypertonicity dramatically increases the osmotic threshold for protein aggregation, demonstrating that protein aggregation-inhibiting pathways are activated by osmotic stress. Our studies demonstrate that hypertonic stress induces protein damage in vivo and that detection and degradation of damaged proteins are essential mechanisms for survival under hypertonic conditions.

scholarly journals Changes in translation rate modulate stress-induced damage of diverse proteins

2013 ◽  
Vol 305 (12) ◽  
pp. C1257-C1264 ◽  
Author(s):  
Heejung Kim ◽  
Kevin Strange
Keyword(s):  
Protein Synthesis ◽  
Protein Aggregation ◽  
Critical Role ◽  
Initiation Factor ◽  
Protein Damage ◽  
Proper Function ◽  
Translation Rate ◽  
Hypertonic Stress ◽  
Endogenous Proteins ◽  
Induced Aggregation

Proteostasis is the maintenance of the proper function of cellular proteins. Hypertonic stress disrupts proteostasis and causes rapid and widespread protein aggregation and misfolding in the nematode Caenorhabditis elegans. Optimal survival in hypertonic environments requires degradation of damaged proteins. Inhibition of protein synthesis occurs in response to diverse environmental stressors and may function in part to minimize stress-induced protein damage. We recently tested this idea directly and demonstrated that translation inhibition by acute exposure to cycloheximide suppresses hypertonicity-induced aggregation of polyglutamine::YFP (Q35::YFP) in body wall muscle cells. In this article, we further characterized the relationship between protein synthesis and hypertonic stress-induced protein damage. We demonstrate that inhibition of translation reduces hypertonic stress-induced formation and growth of Q35::YFP, Q44::YFP, and α-synuclein aggregates; misfolding of paramyosin and ras GTPase; and aggregation of multiple endogenous proteins expressed in diverse cell types. Activation of general control nonderepressible-2 (GCN-2) kinase signaling during hypertonic stress inhibits protein synthesis via phosphorylation of eukaryotic initiation factor-2α (eIF-2α). Inhibition of GCN-2 activation prevents the reduction in translation rate and greatly exacerbates the formation and growth of Q35::YFP aggregates and the aggregation of endogenous proteins. The current studies together with our previous work provide the first direct demonstration that hypertonic stress-induced reduction in protein synthesis minimizes protein aggregation and misfolding. Reduction in translation rate also serves as a signal that activates osmoprotective gene expression. The cellular proteostasis network thus plays a critical role in minimizing hypertonic stress-induced protein damage, in degrading stress-damaged proteins, and in cellular osmosensing and signaling.

scholarly journals Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination

2020 ◽  
Vol 126 (7) ◽  
pp. 907-922 ◽  
Author(s):  
Na Xu ◽  
James Gulick ◽  
Hanna Osinska ◽  
Yang Yu ◽  
Patrick M. McLendon ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
System Performance ◽  
Ubiquitin Proteasome System ◽  
Neonatal Rat ◽  
Autophagic Flux ◽  
Aggregate Formation ◽  
Protein Aggregate ◽  
Ventricular Cardiomyocytes ◽  
A Genome ◽  
Ubiquitin Proteasome

Rationale: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. Objective: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryAB R120G ) and identify how Ube2v1 exerts its effect. Methods and Results: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryAB WT ) or CryAB R120G . Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryAB R120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryAB WT and CryAB R120G -expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryAB R120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1’s function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryAB R120G -induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. Conclusions: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryAB R120G -induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

scholarly journals Optogenetic dissection of mitotic spindle positioning in vivo

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Lars-Eric Fielmich ◽  
Ruben Schmidt ◽  
Daniel J Dickinson ◽  
Bob Goldstein ◽  
Anna Akhmanova ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Membrane Anchor ◽  
Spindle Positioning ◽  
C Elegans ◽  
Cell Cleavage ◽  
Evolutionarily Conserved ◽  
Pulling Forces ◽  
Endogenous Proteins ◽  
Pulling Force

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP–GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

scholarly journals ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

2021 ◽  
Author(s):  
Peng Zhang ◽  
Hai-Yan Qu ◽  
Ziyun Wu ◽  
Huimin Na ◽  
John Hourihan ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Gene Activation ◽  
Proteasome Inhibition ◽  
Ubiquitin Proteasome System ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Proteasomal Inhibition ◽  
New Strategy ◽  
Recovery Response

AbstractThe ubiquitin-proteasome system is vital for cell growth and homeostasis, but for most cancers proteasomal inhibition has not been effective as a therapy. Normal and cancer cells adapt to proteasomal stress through an evolutionarily conserved recovery response, in which the transcription factor NRF1 upregulates proteasome subunit genes. Starting with a C. elegans screen to identify regulators of the recovery response, here we show that this response depends upon phosphorylation of NRF1 on a single residue by the growth factor-activated kinase ERK1/2. Inhibition of this phosphorylation impairs NRF1 nuclear localization and proteasome gene activation, sensitizes C. elegans and cancer cells to proteasomal stress, and synergizes with proteasome inhibition to retard human melanoma growth in vivo in a mouse model. The evolutionarily conserved ERK1/2-NRF1 axis couples proteasome production to growth signaling, and represents a promising new strategy for expanding the range and efficacy of proteasomal inhibition therapy in cancer.

Deciphering the Nature of Caffeic Acid to Inhibit the HSA Aggregation Induced by Glyoxal

2020 ◽  
Vol 27 (8) ◽  
pp. 725-735
Author(s):  
Waseem Feeroze Bhat ◽  
Azaj Ahmed ◽  
Shabeena Abbass ◽  
Mohammad Afsar ◽  
Bilqees Bano ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Caffeic Acid ◽  
Docking Study ◽  
Elevated Concentration ◽  
Protein Aggregate ◽  
Aggregation Effect ◽  
Turbidimetric Analysis ◽  
Induced Aggregation

Background: Under certain circumstances, the path for protein folding deviates and attains an alternative path forming misfolded states, which are the key precursors for protein aggregation. Protein aggregation is associated with variety of diseases and leads to the cytotoxicity. These protein aggregate related diseases have been untreated so far. However, extensive attempts have been applied to develop anti-aggregating agents as possible approaches to overcome protein aggregation. Different types of substances have been reported to halt or decrease the formation of ordered protein aggregates both in vitro and in vivo, such as polyphenols and metal ions. Objective: In the present study the in vitro aggregation of human serum albumin (HSA) by using a reactive dicarbonyl glyoxal has been investigated, simultaneously an attempt has been done to inhibit the glyoxal (GO) induced aggregation of (HSA) by caffeic acid (CA). Methods: Different methods have been employed to investigate the process, fluorescence spectroscopy, circular dichroism, cango red binding assay, thioflavin T dye binding, turbidimetric analysis, docking study and transmission electron microscopy. Results: Results have shown that elevated concentration of GO forms aggregates of HSA, and the activity of CA suggested the possibility of inhibiting the HSA aggregation at higher concentrations, and this compound was found to have an anti-aggregation property. Conclusion: The present study explained that micro molar concentrations of CA inhibits the aggregation of HSA and showed pronounced anti-aggregation effect at increasing concentrations in the presence of GO which is elevated in diabetic and hyperglycaemia conditions.

scholarly journals Tissue-Specific Impact of Autophagy Genes on the Ubiquitin–Proteasome System in C. elegans

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1858 ◽  
Author(s):  
Sweta Jha ◽  
Carina I. Holmberg
Keyword(s):  
Specific Effect ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Tissue Expression ◽  
Compensatory Mechanism ◽  
Tissue Specific ◽  
C Elegans ◽  
Specific Manner ◽  
Ubiquitin Proteasome

The ubiquitin–proteasome system (UPS) and the autophagy–lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.

scholarly journals Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models

2016 ◽  
Vol 11 (1) ◽  
Author(s):  
Matteo Cornaglia ◽  
Gopalan Krishnamani ◽  
Laurent Mouchiroud ◽  
Vincenzo Sorrentino ◽  
Thomas Lehnert ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Neurodegenerative Disease ◽  
C Elegans

scholarly journals Kinetic analysis reveals that independent nucleation events determine the progression of polyglutamine aggregation in C. elegans

2021 ◽  
Vol 118 (11) ◽  
pp. e2021888118
Author(s):  
Tessa Sinnige ◽  
Georg Meisl ◽  
Thomas C. T. Michaels ◽  
Michele Vendruscolo ◽  
Tuomas P. J. Knowles ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Molecular Model ◽  
Kinetic Studies ◽  
Living Organism ◽  
Amyloid Formation ◽  
Complex Environment ◽  
C Elegans ◽  
Wide Range

Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer’s, Parkinson’s, and Huntington’s diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms. Here, we take advantage of the unique properties of a Caenorhabditis elegans model expressing a fluorescently tagged polyglutamine (polyQ) protein, which aggregates into discrete micrometer-sized inclusions that can be directly visualized in real time. We provide a quantitative analysis of protein aggregation in this system and show that the data are described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h−1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic molecular stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results highlight how independent, stochastic nucleation events in individual cells control the overall progression of polyQ aggregation in a living animal. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

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