Protein Misfolding Stress Research Articles

Cancers adapt to their mutational load by buffering protein misfolding stress

In asexual populations that don’t undergo recombination, such as cancer, deleterious mutations are expected to accrue readily due to genome-wide linkage between mutations. Despite this mutational load of often thousands of deleterious mutations, many tumors thrive. How tumors survive the damaging consequences of this mutational load is not well understood. Here, we investigate the functional consequences of mutational load in 10,295 human tumors by quantifying their phenotypic response through changes in gene expression. Using a generalized linear mixed model (GLMM), we find that high mutational load tumors up-regulate proteostasis machinery related to the mitigation and prevention of protein misfolding. We replicate these expression responses in cancer cell lines and show that the viability in high mutational load cancer cells is strongly dependent on complexes that degrade and refold proteins. This indicates that the upregulation of proteostasis machinery is causally important for high mutational burden tumors and uncovers new therapeutic vulnerabilities.

Dysregulation of cholesterol homeostasis is an early signal of β-cell proteotoxicity characteristic of type 2 diabetes.

Type 2 diabetes (T2D) is a common metabolic disease due to insufficient insulin secretion by pancreatic β-cells in the context of insulin resistance. Islet molecular pathology reveals a role for protein misfolding in β-cell dysfunction and loss with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed and cosecreted with insulin. The most toxic form of misfolded IAPP is intracellular membrane disruptive toxic oligomers present in β-cells in T2D and in β-cells of mice transgenic for human IAPP (hIAPP). Prior work revealed a high degree of overlap of transcriptional changes in islets from T2D and prediabetic 9- to 10-wk-old mice transgenic for hIAPP with most changes being pro-survival adaptations and therefore of limited therapeutic guidance. Here, we investigated islets from hIAPP transgenic mice at an earlier age (6 wk) to screen for potential mediators of hIAPP toxicity that precede predominance of pro-survival signaling. We identified early suppression of cholesterol synthesis and trafficking along with aberrant intra-β-cell cholesterol and lipid deposits and impaired cholesterol trafficking to cell membranes. These findings align with comparable lipid deposits present in β-cells in T2D and increased vulnerability to develop T2D in individuals taking medications that suppress cholesterol synthesis.NEW & NOTEWORTHY β-Cell failure in type 2 diabetes (T2D) is characterized by β-cell misfolded protein stress due to the formation of toxic oligomers of islet amyloid polypeptide (IAPP). Most transcriptional changes in islets in T2D are pro-survival adaptations consistent with the slow progression of β-cell loss. In the present study, investigation of the islet transcriptional signatures in a mouse model of T2D expressing human IAPP revealed decreased cholesterol synthesis and trafficking as a plausible early mediator of IAPP toxicity.

Renalase inhibition regulates β cell metabolism to defend against acute and chronic stress.

Renalase (Rnls), annotated as an oxidase enzyme, is a GWAS gene associated with Type 1 Diabetes (T1D) risk. We previously discovered that Rnls inhibition delays diabetes onset in mouse models of T1D in vivo , and protects pancreatic β cells against autoimmune killing, ER and oxidative stress in vitro . The molecular biochemistry and functions of Rnls are entirely uncharted. Here we find that Rnls inhibition defends against loss of β cell mass and islet dysfunction in chronically stressed Akita mice in vivo . We used RNA sequencing, untargeted and targeted metabolomics and metabolic function experiments in mouse and human β cells and discovered a robust and conserved metabolic shift towards glycolysis, amino acid abundance and GSH synthesis to counter protein misfolding stress, in vitro . Our work illustrates a function for Rnls in mammalian cells, and suggests an axis by which manipulating intrinsic properties of β cells can rewire metabolism to protect against diabetogenic stress.

Identification of EGF Receptor and Thrombospondin-1 as Endogenous Targets of ER-Associated Degradation Enhancer EDEM1 in HeLa Cells.

Secretory and membrane proteins are vital for cell activities, including intra- and intercellular communication. Therefore, protein quality control in the endoplasmic reticulum (ER) is an essential and crucial process for eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) targets misfolded proteins during the protein maturation process in the ER and leads to their disposal. This process maintains the ER productive function and prevents misfolded protein stress (i.e., ER stress). The ERAD-stimulating factor ER degradation-enhancing α mannosidase-like 1 protein (EDEM1) acts on misfolded proteins to accelerate ERAD, thereby maintaining the productivity of the ER. However, the detail mechanism underlying the function of EDEM1 in ERAD is not completely understood due to a lack of established physiological substrate proteins. In this study, we attempted to identify substrate proteins for EDEM1 using siRNA. The matrix component thrombospondin-1 (TSP1) and epidermal growth factor receptor (EGFR) were identified as candidate targets of EDEM1. Their protein maturation status and cellular localization were markedly affected by knockdown of EDEM1. We also showed that EDEM1 physically associates with EGFR and enhances EGFR degradation via ERAD. Our data highlight the physiological role of EDEM1 in maintaining specific target proteins and provide a potential approach to the regulation of expression of clinically important proteins.

Graphene Oxide Attenuates Toxicity of Amyloid‐β Aggregates in Yeast by Promoting Disassembly and Boosting Cellular Stress Response

AbstractAlzheimer's disease (AD) is the most prevalent neurodegenerative disease, with the aggregation of misfolded amyloid‐β (Aβ) peptides in the brain being one of its histopathological hallmarks. Recently, graphene oxide (GO) nanoflakes have attracted significant attention in biomedical areas due to their capacity of suppressing Aβ aggregation in vitro. The mechanism of this beneficial effect has not been fully understood in vivo. Herein, the impact of GO on intracellular Aβ42 aggregates and cytotoxicity is investigated using yeast Saccharomyces cerevisiae as the model organism. This study finds that GO nanoflakes can effectively penetrate yeast cells and reduce Aβ42 toxicity. Combination of proteomics data and follow‐up experiments show that GO treatment alters cellular metabolism to increases cellular resistance to misfolded protein stress and oxidative stress, and reduces amounts of intracellular Aβ42 oligomers. Additionally, GO treatment also reduces HTT103QP toxicity in the Huntington's disease (HD) yeast model. The findings offer insights for rationally designing GO nanoflakes‐based therapies for attenuating cytotoxicity of Aβ42, and potentially of other misfolded proteins involved in neurodegenerative pathology.

Identification of protease-sensitive but not misfolding PNLIP variants in familial and hereditary pancreatitis

Mutations in the PNLIP gene have recently been implicated in chronic pancreatitis. Several PNLIP missense variants have been reported to cause protein misfolding and endoplasmic reticulum stress although genetic evidence supporting their association with chronic pancreatitis is currently lacking. Protease-sensitive PNLIP missense variants have also been associated with early-onset chronic pancreatitis although the underlying pathological mechanism remains enigmatic. Herein, we provide new evidence to support the association of protease-sensitive PNLIP variants (but not misfolding PNLIP variants) with pancreatitis. Specifically, we identified protease-sensitive PNLIP variants in 5 of 373 probands (1.3%) with a positive family history of pancreatitis. The protease-sensitive variants, p.F300L and p.I265R, were found to segregate with the disease in three families, including one exhibiting a classical autosomal dominant inheritance pattern. Consistent with previous findings, protease-sensitive variant-positive patients were often characterized by early-onset disease and invariably experienced recurrent acute pancreatitis, although none has so far developed chronic pancreatitis.

Abstract LB075: Cancers adapt to their mutational load by buffering protein misfolding stress

Abstract In asexual populations that don’t undergo recombination, such as cancer, deleterious mutations are expected to accrue readily due to genome-wide linkage between mutations. Despite this mutational load of often thousands of deleterious mutations, many tumors thrive. How tumors survive the damaging consequences of this mutational load is not well understood. Here, we investigate the functional consequences of mutational load in 10,295 human tumors by quantifying their phenotypic response through changes in gene expression. Using a generalized linear mixed model (GLMM), we find that high mutational load tumors up-regulate proteostasis machinery related to the mitigation and prevention of protein misfolding. We replicate these expression responses in cancer cell lines and show that the viability in high mutational load cancer cells is strongly dependent on complexes that degrade and refold proteins. This indicates that upregulation of proteostasis machinery is causally important for high mutational burden tumors and uncovers new therapeutic vulnerabilities. Citation Format: Susanne Tilk, Judith Frydman, Dmitri Petrov, Christina Curtis. Cancers adapt to their mutational load by buffering protein misfolding stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB075.

Tangeretin alleviates Tunicamycin-induced endoplasmic reticulum stress and associated complications in skeletal muscle cells.

Endoplasmic reticulum (ER) stress and associated oxidative stress are involved in the genesis and progression of skeletal muscle diseases such as myositis and atrophy or muscle wasting. Targeting the ER stress and associated downstream pathways can aid in the development of better treatment strategies for these diseases with limited therapeutic approaches. There is a growing interest in identifying natural products against ER stress due to the lower toxicity and cost effectiveness. In the present study, we investigated the protective effect of Tangeretin, a citrus methoxyflavone found in citrus peels against Tunicamycin (pharmacological ER stress inducer)-induced ER stress and associated complications in rat skeletal muscle L6 cell lines. Treatment with Tunicamycin for a period of 24h resulted in the upregulation of ER stress marker proteins, ER resident oxidoreductases and cellular reactive oxygen species (ROS). Co-treatment with Tangeretin was effective in alleviating Tunicamycin-induced ER stress and associated redox-related complications by significantly downregulating the unfolded protein response (UPR), ER resident oxidoreductase proteins, cellular ROS and improving the antioxidant enzyme activity. Tunicamycin also induced upregulation of phosphorylated p38 MAP Kinase and loss of mitochondrial membrane potential. Tangeretin significantly reduced the levels of phosphorylated p38 MAP Kinase and improved the mitochondrial membrane potential. From the results, it is evident that Tangeretin can be explored further as a potential candidate for skeletal muscle diseases involving protein misfolding and ER stress.

The Pseudomonas aeruginosa RpoH (σ32) Regulon and Its Role in Essential Cellular Functions, Starvation Survival, and Antibiotic Tolerance.

The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.

Preclinical mouse model of a misfolded PNLIP variant develops chronic pancreatitis

ObjectiveIncreasing evidence implicates mutation-induced protein misfolding and endoplasm reticulum (ER) stress in the pathophysiology of chronic pancreatitis (CP). The paucity of animal models harbouring genetic risk variants has hampered our...

Neuroprotective effects of intraocular bone marrow‐derived mononuclear cell transplants in inherited retinal degenerations

AbstractInherited retinal degenerations are diseases characterized by photoreceptor loss secondary to genetic defects. At present, only a minority are susceptible of genetic treatment and therefore are a frequent cause of blindness in infancy and adulthood.Retinitis Pigmentosa (RP) is the most frequent inherited retinal degeneration. This disease has a prevalence of around 1 in 4000 individuals and is caused by mutations in more than 300 genes. RP is characterized by progressive loss of photoreceptors, rods and then cones that may die secondarily to rod loss.The rats P23H and Royal college of Surgeon (RCS) are two well characterized animal models of RP because they bear genetic defects observed in RP patients. The P23H rat suffers an autosomal dominant mutation of the rhodopsin gene that causes protein misfolding and endoplasmic reticulum stress in rods, which die first followed by cones. The RCS rat has a mutation of MERTK gene, that impedes the normal photoreceptor outer segment phagocytosis by RPE cells thus causing simultaneous rod and cone degeneration.Therapies for inherited photoreceptor degeneration aim to prevent or delay death of the photoreceptors, or in specific circumstances try to replace them.The Bone marrow derived‐mononuclear cells (BM‐MNCs) are a mononuclear CD34+ subpopulation of bone marrow‐derived cells that can be separated from the other cellular fractions of the bone marrow aspirate using a Ficoll density gradient‐based protocol. The BM‐MNCs is a mixture of specific bone marrow cell subtypes in which is possible to find cells: Lin+, Lin−, CD34+, c‐Kit+, CD133+ or Sca‐1+ HSCs, endothelial precursor cells, and Mesenchymal Stem Cells.BM‐MNCs transplantation has shown promising results in different animal models of neuronal degeneration, including inherited photoreceptor degenerations.In our studies we have performed a syngeneic intravitreal or subretinal transplantation of BM‐MNCs in a P23H‐1 and RCS rats, and we have documented that both increase photoreceptor survival. They also improve the morphology of the photoreceptors outer segment, decrease synaptic degeneration in the outer plexiform layer and reduce glial fibrillary acidic protein (GFAP) expression in Müller cells. However, the transplanted cells did not penetrate the retina, only survived for up to 15 days, and did not modify microglial cell activation and migration or the electroretinographic responses. Their beneficial effects are maybe due to a paracrine trophic effect achieved through release of neurotrophic, anti‐angiogenic and/or immunomodulatory factors.In conclusion, the intra ocular transplantation BM‐MNCs shows photoreceptor neuroprotective actions in inherited retinal degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore could be indicated for treatment of the early stages of photoreceptor degenerations.

Impact of Environmental Risk Factors on Mitochondrial Dysfunction, Neuroinflammation, Protein Misfolding, and Oxidative Stress in the Etiopathogenesis of Parkinson's Disease.

As a prevalent progressive neurodegenerative disorder, Parkinson’s disease (PD) is characterized by the neuropathological hallmark of the loss of nigrostriatal dopaminergic (DAergic) innervation and the appearance of Lewy bodies with aggregated α-synuclein. Although several familial forms of PD have been reported to be associated with several gene variants, most cases in nature are sporadic, triggered by a complex interplay of genetic and environmental risk factors. Numerous epidemiological studies during the past two decades have shown positive associations between PD and several environmental factors, including exposure to neurotoxic pesticides/herbicides and heavy metals as well as traumatic brain injury. Other environmental factors that have been implicated as potential risk factors for PD include industrial chemicals, wood pulp mills, farming, well-water consumption, and rural residence. In this review, we summarize the environmental toxicology of PD with the focus on the elaboration of chemical toxicity and the underlying pathogenic mechanisms associated with exposure to several neurotoxic chemicals, specifically 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat (PQ), dichloro-diphenyl-trichloroethane (DDT), dieldrin, manganese (Mn), and vanadium (V). Our overview of the current findings from cellular, animal, and human studies of PD provides information for possible intervention strategies aimed at halting the initiation and exacerbation of environmentally linked PD.

PET- and ICT-Based Ratiometric Probe: An Unusual Phenomenon of Morpholine-Conjugated Fluorophore for Mitochondrial pH Mapping during Mitophagy

Mitochondrial functions are heavily influenced by acid-base homeostasis. Hence, elucidation of the mitochondrial pH is essential in living cells, and its alterations during pathologies is an interesting question to be addressed. Small molecular fluorescent probes are progressively applied to quantify the mitochondrial pH by fluorescence imaging. Herein, we designed a unique small molecular fluorescent probe, PM-Mor-OH, based on the lipophilic morpholine ligand-conjugated pyridinium derivative of "IndiFluors". The morpholine-conjugated fluorescent probe usually localized the lysosome. However, herein, we observed unusual phenomena of morpholine-tagged PM-Mor-OH that localized mitochondria explicitly. The morpholine ligand also plays a pivotal role in tuning optical properties via photoinduced electron transfer (PET) during internal pH alteration (ΔpHi). In the mitophagy process, lysosomes engulf damaged mitochondria, leading to ΔpHi, which can be monitored using our probe. It exhibited "ratiometric" emission at single wavelength excitation (ex. 488) and is suitable for monitoring and quantifying the ΔpHi using confocal microscope high-resolution image analysis during mitophagy. The bathochromic emission shifts due to intramolecular charge transfer (ICT) in basic pH were well explained by the time-dependent density functional theory (TD-DFT/PCM). Similarly, the change in the emission ratio (green/red) with pH variations was also validated by the PET process. In addition, PM-Mor-OH can quantify the pH change during oxidative stress induced by rapamycin, mutant A53T α-synuclein-mediated protein misfolding stress in mitochondria, and during starvation. Rapamycin-induced mitophagy was further elucidated by the translocation of mCherry Parkin to damaged mitochondria, which well correlates with our probe. Thus, PM-Mito-OH is a valuable probe for visualizing mitophagy and can act as a suitable tool for the diagnosis of mitochondrial diseases.

The unfolded protein response transducer IRE1α promotes reticulophagy in podocytes

Glomerular diseases involving podocyte/glomerular epithelial cell (GEC) injury feature protein misfolding and endoplasmic reticulum (ER) stress. Inositol-requiring enzyme 1α (IRE1α) mediates chaperone production and autophagy during ER stress. We examined the role of IRE1α in selective autophagy of the ER (reticulophagy). Control and IRE1α knockout (KO) GECs were incubated with tunicamycin to induce ER stress and subjected to proteomic analysis. This showed IRE1α-dependent upregulation of secretory pathway mediators, including the coat protein complex II component Sec23B. Tunicamycin enhanced expression of Sec23B and the reticulophagy adaptor reticulon-3-long (RTN3L) in control, but not IRE1α KO GECs. Knockdown of Sec23B reduced autophagosome formation in response to ER stress. Tunicamycin stimulated colocalization of autophagosomes with Sec23B and RTN3L in an IRE1α-dependent manner. Similarly, during ER stress, glomerular α5 collagen IV colocalized with RTN3L and autophagosomes. Degradation of RTN3L and collagen IV increased in response to tunicamycin, and the turnover was blocked by deletion of IRE1α; thus, the IRE1α pathway promotes RTN3L-mediated reticulophagy and collagen IV may be an IRE1α-dependent reticulophagy substrate. In experimental glomerulonephritis, expression of Sec23B, RTN3L, and LC3-II increased in glomeruli of control mice, but not in podocyte-specific IRE1α KO littermates. In conclusion, during ER stress, IRE1α redirects a subset of Sec23B-positive vesicles to deliver RTN3L-coated ER fragments to autophagosomes. Reticulophagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.

Application of ToxTracker for the toxicological assessment of tobacco and nicotine delivery products

Consumer demands and innovation have led to an increasingly diverse range of nicotine delivery systems, driven by a desire to reduce risk associated with traditional combustible cigarettes. This speed of change provides a mandate for rapid new product assessment. We have used the validated technology ToxTracker®, to assess biomarkers of DNA damage, protein misfolding, oxidative and cellular stress, across the categories of cigarette (1R6F), tobacco heating product (THP 1.4) and electronic cigarette (ePen 3). In addition, we compared the commonly used test matrices for tobacco and nicotine products; whole aerosol aqueous extracts (AqE) and gas vapour phase (GVP), determining their suitability across the product categories.We demonstrated a significant reduction in oxidative stress and cytotoxicity for THP 1.4 over cigarette, further reduced for ePen 3, when assessed by both dilution and nicotine dosimetry. We also identified that while the extraction matrices AqE and GVP from combustible products were equivalent in the induced responses, this was not true of the other category examples, moreover THP 1.4 GVP demonstrates a >50 % reduction in both toxicity and cytotoxicity endpoints over AqE. This indicates that unlike cigarette, the active components or toxicants for THP and electronic cigarette are associated with the aerosol fraction of these categories.

Pathological β-Cell Endoplasmic Reticulum Stress in Type 2 Diabetes: Current Evidence.

The notion that in diabetes pancreatic β-cells express endoplasmic reticulum (ER) stress markers indicative of increased unfolded protein response (UPR) signaling is no longer in doubt. However, what remains controversial is whether this increase in ER stress response actually contributes importantly to the β-cell failure of type 2 diabetes (akin to ‘terminal UPR’), or whether it represents a coping mechanism that represents the best attempt of β-cells to adapt to changes in metabolic demands as presented by disease progression. Here an intercontinental group of experts review evidence for the role of ER stress in monogenic and type 2 diabetes in an attempt to reconcile these disparate views. Current evidence implies that pancreatic β-cells require a regulated UPR for their development, function and survival, as well as to maintain cellular homeostasis in response to protein misfolding stress. Prolonged ER stress signaling, however, can be detrimental to β-cells, highlighting the importance of “optimal” UPR for ER homeostasis, β-cell function and survival.

MARK2 phosphorylates eIF2α in response to proteotoxic stress.

The regulation of protein synthesis is essential for maintaining cellular homeostasis, especially during stress responses, and its dysregulation could underlie the development of human diseases. The critical step during translation regulation is the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α). Here we report the identification of a direct kinase of eIF2α, microtubule affinity-regulating kinase 2 (MARK2), which phosphorylates eIF2α in response to proteotoxic stress. The activity of MARK2 was confirmed in the cells lacking the 4 previously known eIF2α kinases. MARK2 itself was found to be a substrate of protein kinase C delta (PKCδ), which serves as a sensor for protein misfolding stress through a dynamic interaction with heat shock protein 90 (HSP90). Both MARK2 and PKCδ are activated via phosphorylation in proteotoxicity-associated neurodegenerative mouse models and in human patients with amyotrophic lateral sclerosis (ALS). These results reveal a PKCδ-MARK2-eIF2α cascade that may play a critical role in cellular proteotoxic stress responses and human diseases.

Tolerance to nascent protein misfolding stress requires fine-tuning of the cAMP/PKA pathway

Protein aggregation is the abnormal association of misfolded proteins into larger, often insoluble structures that can be toxic during aging and in protein aggregation-associated diseases. Previous research has established a role for the cytosolic Tsa1 peroxiredoxin in responding to protein misfolding stress. Tsa1 is also known to downregulate the cAMP/protein kinase A (PKA) pathway as part of the response to hydrogen peroxide stress. However, whether the cAMP/PKA pathway is involved in protein misfolding stress is not known. Using transcriptomics, we examined the response to protein misfolding stress and found upregulation of numerous stress gene functions and downregulation of many genes related to protein synthesis and other growth-related processes consistent with the well-characterized environmental stress response. The scope of the transcriptional response is largely similar in wild-type and tsa1 mutant strains, but the magnitude is dampened in the strain lacking Tsa1. We identified a direct protein interaction between Tsa1 and the Bcy1 regulatory subunit of PKA that is present under normal growth conditions and explains the observed differences in gene expression profiles. This interaction is increased in a redox-dependent manner in response to nascent protein misfolding, via Tsa1-mediated oxidation of Bcy1. Oxidation of Bcy1 causes a reduction in cAMP binding by Bcy1, which dampens PKA pathway activity, leading to a targeted reprogramming of gene expression. Redox regulation of the regulatory subunit of PKA provides a mechanism to mitigate the toxic consequences of protein misfolding stress that is distinct to stress caused by exogenous sources of reactive oxygen species.

Interspecies Differences in Proteome Turnover Kinetics Are Correlated With Life Spans and Energetic Demands

AbstractCells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover, and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse life spans including the longest-lived mammal, the bowhead whale. We show that organismal life span is negatively correlated with turnover rates of highly abundant proteins. In comparison with mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production, and reduced reactive oxygen species levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of a rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

4E-BP1 Protects Neurons from Misfolded Protein Stress and Parkinson's Disease Toxicity by Inducing the Mitochondrial Unfolded Protein Response.

Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4E-BP1 is a key node in the regulation of protein synthesis, as activated 4E-BP1 represses global protein translation. Overexpression of 4E-BP1 mediates the benefits of dietary restriction and can counter metabolic stress, and 4E-BP1 disinhibition on mTORC1 repression may be neuroprotective; however, whether 4E-BP1 overexpression is neuroprotective in mammalian neurons is yet to be fully explored. To address this question, we generated 4E-BP1-overexpressing transgenic mice and confirmed marked reductions in protein translation in 4E-BP1-overexpressing primary neurons. After documenting that 4E-BP1-overexpressing neurons are resistant to proteotoxic stress elicited by brefeldin A treatment, we exposed primary neurons to three different Parkinson's disease (PD)-linked toxins (rotenone, maneb, or paraquat) and documented significant protection in neurons from newborn male and female 4E-BP1-OE transgenic mice. We observed 4E-BP1-dependent upregulation of genes encoding proteins that comprise the mitochondrial unfolded protein response, and noted 4E-BP1 overexpression required activation of the mitochondrial unfolded protein response for neuroprotection against rotenone toxicity. We also tested whether 4E-BP1 could prevent α-synuclein neurotoxicity by treating 4E-BP1-overexpressing primary neurons with α-synuclein preformed fibrils, and we observed marked reductions in α-synuclein aggregation and neurotoxicity, thus validating that 4E-BP1 is a powerful suppressor of PD-linked pathogenic insults. Our results indicate that increasing 4E-BP1 expression or enhancing 4E-BP1 activation can robustly induce the mitochondrial unfolded protein response and thus could be an appealing strategy for treating a variety of neurodegenerative diseases, including especially PD.SIGNIFICANCE STATEMENT In neurodegenerative disease, misfolded proteins accumulate and overwhelm normal systems of homeostasis and quality control. One mechanism for improving protein quality control is to reduce protein translation. Here we investigated whether neuronal overexpression of 4E-BP1, a key repressor of protein translation, can protect against misfolded protein stress and toxicities linked to Parkinson's disease, and found that 4E-BP1 overexpression prevented cell death in neurons treated with brefeldin A, rotenone, maneb, paraquat, or preformed fibrils of α-synuclein. When we sought the basis for 4E-BP1 neuroprotection, we discovered that 4E-BP1 activation promoted the mitochondrial unfolded protein response. Our findings highlight 4E-BP1 as a therapeutic target in neurodegenerative disease and underscore the importance of the mitochondrial unfolded protein response in neuroprotection against various insults.