bims-ershed Biomed News
on ER Stress in Health and Diseases
Issue of 2022‒01‒23
six papers selected by
Matías Eduardo González Quiroz
Worker’s Hospital

  1. Anim Cells Syst (Seoul). 2021 ;25(6): 347-357
      The endoplasmic reticulum (ER) can sense a wide variety of external and internal perturbations and responds by mounting stress coping responses, such as the unfolded protein response (UPR). The UPR is composed of three stress sensors, namely IRE1α, PERK, and ATF6 that are activated to re-establish ER homeostasis. IRE1α represents the most ancient branch of the UPR affecting many cellular processes in plant and animal cells. IRE1α is a type I transmembrane protein with kinase/nuclease activities in response to ER stress. Both the ER luminal and cytosolic IRE1α interactomes have been identified revealing a multifunctional role of the ER stress sensor. IRE1α is also associated with organellar membrane contacts to promote rapid communication between intracellular organelles under stress conditions.
    Keywords:  IRE1α; endoplasmic reticulum; stress sensor; unfolded protein response
  2. Cell Biol Toxicol. 2022 Jan 18.
      Covalent binding of reactive metabolites formed by drug metabolic activation with biological macromolecules is considered to be an important mechanism of drug metabolic toxicity. Recent studies indicate that the endoplasmic reticulum (ER) could play an important role in drug toxicity by participating in the metabolic activation of drugs and could be a primarily attacked target by reactive metabolites. In this article, we summarize the generation and mechanism of reactive metabolites in ER stress and their associated cell death and inflammatory cascade, as well as the systematic modulation of unfolded protein response (UPR)-mediated adaptive pathways.
    Keywords:  Apoptosis; Drug metabolic toxicity; Endoplasmic reticulum; Inflammation; Reactive metabolites; Unfolded protein response
  3. Acc Chem Res. 2022 Jan 18.
      ConspectusProtein aggregation is a biological phenomenon in which aberrantly processed or mutant proteins misfold and assemble into a variety of insoluble aggregates. Decades of studies have delineated the structure, interaction, and activity of proteins in either their natively folded structures or insoluble aggregates such as amyloid fibrils. However, a variety of intermediate species exist between these two extreme states in the protein folding landscape. Herein, we collectively term these intermediate species as misfolded protein oligomers, including soluble oligomers and preamyloid oligomers that are formed by unfolded or misfolded proteins. While extensive tools have been developed to study folded proteins or amyloid fibrils, research to understand the properties and activities of misfolded protein oligomers has been limited by the lack of methods to detect and interrogate these species in live cells.In this Account, we describe our efforts in the development of chemical methods that allow for the characterization of the multistep protein aggregation process, in particular the misfolded protein oligomers, in living cells. As the start of this journey, we attempted to develop a fluorogenic method wherein the misfolded oligomers could turn on the fluorescence of chemical probes that are conjugated to the protein-of-interest (POI). To this end, we produced a series of destabilized HaloTag variants, formulating the primary component of the AgHalo sensor, which misfolds and aggregates when cells are subjected to stress. When AgHalo is covalently conjugated with a solvatochromic fluorophore, misfolding of the AgHalo conjugate would activate fluorescence, resulting in the observation of misfolded oligomers. Following this work, we extended the scope of detection from AgHalo to any protein-of-interest via the AggTag method, wherein the POIs are genetically fused to self-labeling protein tags (HaloTag or SNAP-tag). Focusing on the molecular rotor-based fluorophores, we applied the modulated fluorescent protein (FP) chromophore core as a prototype for the AggTag probes, to enable the fluorogenic detection of misfolded soluble oligomers of multiple proteins in live cells. Next, we further developed the AggTag method to distinguish insoluble aggregates from misfolded oligomers, using two classes of probes that activate different fluorescence emission toward these two conformations. To enable this goal, we applied physical organic chemistry and computational chemistry to discover a new category of triode-like fluorophores, wherein the π orbitals of either an electron density regulator or the donor-acceptor linkages are used to control the rotational barriers of fluorophores in the excited states. This mechanism allows us to rationally design molecular rotor-based fluorophores that have desired responses to viscosity, thus extending the application of the AggTag method.In summary, our work allows the direct monitoring of the misfolded protein oligomers and differentiation of insoluble aggregates from other conformations in live cells, thus enabling studies of many currently unanswered questions in protein aggregation. Future directions are to develop methods that enable quantitative analyses of the protein aggregation process. Further, new methods are needed to detect and to quantify the formation and maturation of protein or RNA condensates that form membraneless organelles.
  4. Front Physiol. 2021 ;12 793171
      Atrial fibrillation (AF) remains the most common arrhythmia seen clinically. The incidence of AF is increasing due to the aging population. AF is associated with a significant increase in morbidity and mortality, yet current treatment paradigms have proven largely inadequate. Therefore, there is an urgent need to develop new effective therapeutic strategies for AF. The endoplasmic reticulum (ER) in the heart plays critical roles in the regulation of excitation-contraction coupling and cardiac function. Perturbation in the ER homeostasis due to intrinsic and extrinsic factors, such as inflammation, oxidative stress, and ischemia, leads to ER stress that has been linked to multiple conditions including diabetes mellitus, neurodegeneration, cancer, heart disease, and cardiac arrhythmias. Recent studies have documented the critical roles of ER stress in the pathophysiological basis of AF. Using an animal model of chronic pressure overload, we demonstrate a significant increase in ER stress in atrial tissues. Moreover, we demonstrate that treatment with a small molecule inhibitor to inhibit the soluble epoxide hydrolase enzyme in the arachidonic acid metabolism significantly reduces ER stress as well as atrial electrical and structural remodeling. The current review article will attempt to provide a perspective on our recent understandings and current knowledge gaps on the critical roles of proteostasis and ER stress in AF progression.
    Keywords:  atrial fibrillation; electrical remodeling; endoplasmic reticulum stress; inflammation; oxidative stress; structural remodeling
  5. Nature. 2022 Jan 19.
      Ageing is accompanied by a decline in cellular proteostasis, which underlies many age-related protein misfolding diseases1,2. Yet, how ageing impairs proteostasis remains unclear. As nascent polypeptides represent a substantial burden on the proteostasis network3, we hypothesized that altered translational efficiency during ageing could help to drive the collapse of proteostasis. Here we show that ageing alters the kinetics of translation elongation in both Caenorhabditis elegans and Saccharomyces cerevisiae. Ribosome pausing was exacerbated at specific positions in aged yeast and worms, including polybasic stretches, leading to increased ribosome collisions known to trigger ribosome-associated quality control (RQC)4-6. Notably, aged yeast cells exhibited impaired clearance and increased aggregation of RQC substrates, indicating that ageing overwhelms this pathway. Indeed, long-lived yeast mutants reduced age-dependent ribosome pausing, and extended lifespan correlated with greater flux through the RQC pathway. Further linking altered translation to proteostasis collapse, we found that nascent polypeptides exhibiting age-dependent ribosome pausing in C. elegans were strongly enriched among age-dependent protein aggregates. Notably, ageing increased the pausing and aggregation of many components of proteostasis, which could initiate a cycle of proteostasis collapse. We propose that increased ribosome pausing, leading to RQC overload and nascent polypeptide aggregation, critically contributes to proteostasis impairment and systemic decline during ageing.
  6. Curr Protoc. 2022 Jan;2(1): e344
      The control of mRNA stability is fundamental to gene regulation, and a deeper understanding of this post-transcriptional regulatory step can provide key insights into gene function. Measuring mRNA half-lives directly, however, is challenging. The most common strategies for evaluating mRNA stability and decay involve blocking general transcription and then measuring the decline in mRNA levels over time. The downside of these approaches, however, is that they severely impact cell function and viability, indirectly perturbing gene expression. Here, we describe Roadblock-qPCR, a simple method for measuring mRNA decay kinetics in living cells that is both economical and quick. Cells are first incubated with the nucleoside analog 4-thiouridine (4sU), which is readily incorporated into nascent mRNAs during transcription. RNA is then extracted and treated with N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that selectively modifies 4sU, before proceeding to cDNA synthesis. Because the NEM-modified 4sU creates a chemical "roadblock" that interferes with reverse transcription, this treatment ultimately results in the depletion of the nascent 4sU-containing transcripts from the cDNA pool. As such, the decay rate of the non-4sU-labeled pre-existing mRNAs can be monitored by quantitative PCR (qPCR). In combination with spike-in standards, this approach can be used to efficiently and accurately measure the half-lives of endogenous mRNAs with a wide range of stabilities, while avoiding the artifacts of transcription shutoff strategies. © 2022 Wiley Periodicals LLC. Basic Protocol: Roadblock-qPCR Support Protocol: Synthesis of spike-in mRNA.
    Keywords:  4-thiouridine; mRNA stability; qPCR