Senataxin deficiency disrupts proteostasis through nucleolar ncRNA-driven protein aggregation.

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.

Bax Inhibitor-1 preserves pancreatic ß-cell proteostasis by limiting proinsulin misfolding and programmed cell death.

The prevalence of diabetes steadily increases worldwide mirroring the prevalence of obesity. Endoplasmic reticulum (ER) stress is activated in diabetes and contributes to ß-cell dysfunction and apoptosis through the activation of a terminal unfolded protein response (UPR). Our results uncover a new role for Bax Inhibitor-One (BI-1), a negative regulator of inositol-requiring enzyme 1 (IRE1α) in preserving ß-cell health against terminal UPR-induced apoptosis and pyroptosis in the context of supraphysiological loads of insulin production. BI-1-deficient mice experience a decline in endocrine pancreatic function in physiological and pathophysiological conditions, namely obesity induced by high-fat diet (HFD). We observed early-onset diabetes characterized by hyperglycemia, reduced serum insulin levels, ß-cell loss, increased pancreatic lipases and pro-inflammatory cytokines, and the progression of metabolic dysfunction. Pancreatic section analysis revealed that BI-1 deletion overburdens unfolded proinsulin in the ER of ß-cells, confirmed by ultrastructural signs of ER stress with overwhelmed IRE1α endoribonuclease (RNase) activity in freshly isolated islets. ER stress led to ß-cell dysfunction and islet loss, due to an increase in immature proinsulin granules and defects in insulin crystallization with the presence of Rod-like granules. These results correlated with the induction of autophagy, ER phagy, and crinophagy quality control mechanisms, likely to alleviate the atypical accumulation of misfolded proinsulin in the ER. In fine, BI-1 in ß-cells limited IRE1α RNase activity from triggering programmed ß-cell death through apoptosis and pyroptosis (caspase-1, IL-1ß) via NLRP3 inflammasome activation and metabolic dysfunction. Pharmaceutical IRE1α inhibition with STF-083010 reversed ß-cell failure and normalized the metabolic phenotype. These results uncover a new protective role for BI-1 in pancreatic ß-cell physiology as a stress integrator to modulate the UPR triggered by accumulating unfolded proinsulin in the ER, as well as autophagy and programmed cell death, with consequences on ß-cell function and insulin secretion. In pancreatic ß-cells, BI-1-/- deficiency perturbs proteostasis with proinsulin misfolding, ER stress, terminal UPR with overwhelmed IRE1α/XBP1s/CHOP activation, inflammation, ß-cell programmed cell death, and diabetes.

The role of labile iron on brain proteostasis; could it be an early event of neurodegenerative disease?

Iron deposits in the brain are a natural consequence of aging. Iron accumulation, especially in the form of labile iron, can trigger a cascade of adverse effects, eventually leading to neurodegeneration and cognitive decline. Aging also increases the dysfunction of cellular proteostasis. The question of whether iron alters proteostasis is now being pondered. Herein, we investigated the effect of ferric citrate, considered as labile iron, on various aspects of proteostasis of neuronal cell lines, and also established an animal model having a labile iron diet in order to evaluate proteostasis alteration in the brain along with behavioral effects. According to an in vitro study, labile iron was found to activate lysosome formation but inhibits lysosomal clearance function. Furthermore, the presence of labile iron can alter autophagic flux and can also induce the accumulation of protein aggregates. RNA-sequencing analysis further reveals the upregulation of various terms related to proteostasis along with neurodegenerative disease-related terms. According to an in vivo study, a labile iron-rich diet does not induce iron overload conditions and was not detrimental to the behavior of male Wistar rats. However, an iron-rich diet can promote iron accumulation in a region-dependent manner. By staining for autophagic markers and misfolding proteins in the cerebral cortex and hippocampus, an iron-rich diet was actually found to alter autophagy and induce an accumulation of misfolding proteins. These findings emphasize the importance of labile iron on brain cell proteostasis, which could be implicated in developing of neurological diseases.

Tracing genetic diversity captures the molecular basis of misfolding disease.

Genetic variation in human populations can result in the misfolding and aggregation of proteins, giving rise to systemic and neurodegenerative diseases that require management by proteostasis. Here, we define the role of GRP94, the endoplasmic reticulum Hsp90 chaperone paralog, in managing alpha-1-antitrypsin deficiency on a residue-by-residue basis using Gaussian process regression-based machine learning to profile the spatial covariance relationships that dictate protein folding arising from sequence variants in the population. Covariance analysis suggests a role for the ATPase activity of GRP94 in controlling the N- to C-terminal cooperative folding of alpha-1-antitrypsin responsible for the correction of liver aggregation and lung-disease phenotypes of alpha-1-antitrypsin deficiency. Gaussian process-based spatial covariance profiling provides a standard model built on covariant principles to evaluate the role of proteostasis components in guiding information flow from genome to proteome in response to genetic variation, potentially allowing us to intervene in the onset and progression of complex multi-system human diseases.

Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy.

Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.

Proteostatic Remodeling of Small Heat Shock Chaperones─Crystallins by Ran-Binding Protein 2─and the Peptidyl-Prolyl cis-trans Isomerase and Chaperone Activities of Its Cyclophilin Domain.

Disturbances in protein phase transitions promote protein aggregation─a neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also regulate phase transitions and proteostasis by suppressing protein aggregation. Ranbp2 haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against phototoxicity by proteostatic regulations of neuroprotective substrates of Ranbp2 and by suppressing the buildup of polyubiquitylated substrates. Losses of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 recapitulate molecular effects of Ranbp2 haploinsufficiency. These CY impairments also stimulate deubiquitylation activities and phase transitions of 19S cap subunits of the 26S proteasome that associates with Ranbp2. However, links between CY moonlighting activity, substrate ubiquitylation, and proteostasis remain incomplete. Here, we reveal the Ranbp2 regulation of small heat shock chaperones─crystallins in the chorioretina by proteomics of mice with total or selective modular deficits of Ranbp2. Specifically, loss of CY PPIase of Ranbp2 upregulates αA-Crystallin, which is repressed in adult nonlenticular tissues. Conversely, impairment of CY's chaperone activity opposite to the PPIase pocket downregulates a subset of αA-Crystallin's substrates, γ-crystallins. These CY-dependent effects cause age-dependent and chorioretinal-selective declines of ubiquitylated substrates without affecting the chorioretinal morphology. A model emerges whereby inhibition of Ranbp2's CY PPIase remodels crystallins' expressions, subdues molecular aging, and preordains the chorioretina to neuroprotection by augmenting the chaperone capacity and the degradation of polyubiquitylated substrates against proteostatic impairments. Further, the druggable Ranbp2 CY holds pan-therapeutic potential against proteotoxicity and neurodegeneration.

Alterations in Proteostasis Mechanisms in Niemann-Pick Type C Disease.

Niemann-Pick Type C (NPC) represents an autosomal recessive disorder with an incidence rate of 1 in 150,000 live births, classified within lysosomal storage diseases (LSDs). The abnormal accumulation of unesterified cholesterol characterizes the pathophysiology of NPC. This phenomenon is not unique to NPC, as analogous accumulations have also been observed in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. Interestingly, disturbances in the folding of the mutant protein NPC1 I1061T are accompanied by the aggregation of proteins such as hyperphosphorylated tau, α-synuclein, TDP-43, and ß-amyloid peptide. These accumulations suggest potential disruptions in proteostasis, a regulatory process encompassing four principal mechanisms: synthesis, folding, maintenance of folding, and protein degradation. The dysregulation of these processes leads to excessive accumulation of abnormal proteins that impair cell function and trigger cytotoxicity. This comprehensive review delineates reported alterations across proteostasis mechanisms in NPC, encompassing changes in processes from synthesis to degradation. Additionally, it discusses therapeutic interventions targeting pharmacological facets of proteostasis in NPC. Noteworthy among these interventions is valproic acid, a histone deacetylase inhibitor (HDACi) that modulates acetylation during NPC1 synthesis. In addition, various therapeutic options addressing protein folding modulation, such as abiraterone acetate, DHBP, calnexin, and arimoclomol, are examined. Additionally, treatments impeding NPC1 degradation, exemplified by bortezomib and MG132, are explored as potential strategies. This review consolidates current knowledge on proteostasis dysregulation in NPC and underscores the therapeutic landscape targeting diverse facets of this intricate process.

VCP/p97 mediates nuclear targeting of non-ER-imported prion protein to maintain proteostasis.

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.

Age-progressive interplay of HSP-proteostasis, ECM-cell junctions and biomechanics ensures C. elegans astroglial architecture.

Tissue integrity is sensitive to temperature, tension, age, and is sustained throughout life by adaptive cell-autonomous or extrinsic mechanisms. Safeguarding the remarkably-complex architectures of neurons and glia ensures age-dependent integrity of functional circuits. Here, we report mechanisms sustaining the integrity of C. elegans CEPsh astrocyte-like glia. We combine large-scale genetics with manipulation of genes, cells, and their environment, quantitative imaging of cellular/ subcellular features, tissue material properties and extracellular matrix (ECM). We identify mutants with age-progressive, environment-dependent defects in glial architecture, consequent disruption of neuronal architecture, and abnormal aging. Functional loss of epithelial Hsp70/Hsc70-cochaperone BAG2 causes ECM disruption, altered tissue biomechanics, and hypersensitivity of glia to environmental temperature and mechanics. Glial-cell junctions ensure epithelia-ECM-CEPsh glia association. Modifying glial junctions or ECM mechanics safeguards glial integrity against disrupted BAG2-proteostasis. Overall, we present a finely-regulated interplay of proteostasis-ECM and cell junctions with conserved components that ensures age-progressive robustness of glial architecture.

Role of autophagy and proteostasis in neurodegenerative diseases: Exploring the therapeutic interventions.

Neurodegenerative disorders are devastating disorders characterized by gradual loss of neurons and cognition or mobility impairment. The common pathological features of these diseases are associated with the accumulation of misfolded or aggregation of proteins. The pivotal roles of autophagy and proteostasis in maintaining cellular health and preventing the accumulation of misfolded proteins, which are associated with neurodegenerative diseases like Huntington's disease (HD), Alzheimer's disease (AD), and Parkinson's disease (PD). This article presents an in-depth examination of the interplay between autophagy and proteostasis, highlighting how these processes cooperatively contribute to cellular homeostasis and prevent pathogenic protein aggregate accumulation. Furthermore, the review emphasises the potential therapeutic implications of targeting autophagy and proteostasis to mitigate neurodegenerative diseases. While advancements in research hold promise for developing novel treatments, the article also addresses the challenges and complexities associated with modulating these intricate cellular pathways. Ultimately, advancing understanding of the underlying mechanism of autophagy and proteostasis in neurodegenerative disorders provides valuable insights into potential therapeutic avenues and future research directions.

Maximum entropy determination of mammalian proteome dynamics.

Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

Aging in Heart Failure: Embracing Biology Over Chronology: JACC Family Series.

Age is among the most potent risk factors for developing heart failure and is strongly associated with adverse outcomes. As the global population continues to age and the prevalence of heart failure rises, understanding the role of aging in the development and progression of this chronic disease is essential. Although chronologic age is on a fixed course, biological aging is more variable and potentially modifiable in patients with heart failure. This review describes the current knowledge on mechanisms of biological aging that contribute to the pathogenesis of heart failure. The discussion focuses on 3 hallmarks of aging-impaired proteostasis, mitochondrial dysfunction, and deregulated nutrient sensing-that are currently being targeted in therapeutic development for older adults with heart failure. In assessing existing and emerging therapeutic strategies, the review also enumerates the importance of incorporating geriatric conditions into the management of older adults with heart failure and in ongoing clinical trials.

Involvement of the glymphatic/meningeal lymphatic system in Alzheimer's disease: insights into proteostasis and future directions.

BACKGROUND:  Alzheimer's disease (AD) is pathologically characterized by the abnormal accumulation of Aß and tau proteins. There has long been a keen interest among researchers in understanding how Aß and tau are ultimately cleared in the brain. The discovery of this glymphatic system introduced a novel perspective on protein clearance and it gained recognition as one of the major brain clearance pathways for clearing these pathogenic proteins in AD. This finding has sparked interest in exploring the potential contribution of the glymphatic/meningeal lymphatic system in AD. Furthermore, there is a growing emphasis and discussion regarding the possibility that activating the glymphatic/meningeal lymphatic system could serve as a novel therapeutic strategy against AD. OBJECTIVES:  Given this current research trend, the primary focus of this comprehensive review is to highlight the role of the glymphatic/meningeal lymphatic system in the pathogenesis of AD. The discussion will encompass future research directions and prospects for treatment in relation to the glymphatic/meningeal lymphatic system.

Adenosine Triphosphate: The Primordial Molecule That Controls Protein Homeostasis and Shapes the Genome-Proteome Interface.

Adenosine triphosphate (ATP) acts as the universal energy currency that drives various biological processes, while nucleic acids function to store and transmit genetic information for all living organisms. Liquid-liquid phase separation (LLPS) represents the common principle for the formation of membrane-less organelles (MLOs) composed of proteins rich in intrinsically disordered regions (IDRs) and nucleic acids. Currently, while IDRs are well recognized to facilitate LLPS through dynamic and multivalent interactions, the precise mechanisms by which ATP and nucleic acids affect LLPS still remain elusive. This review summarizes recent NMR results on the LLPS of human FUS, TDP-43, and the viral nucleocapsid (N) protein of SARS-CoV-2, as modulated by ATP and nucleic acids, revealing the following: (1) ATP binds to folded domains overlapping with nucleic-acid-binding interfaces; (2) ATP and nucleic acids interplay to biphasically modulate LLPS by competitively binding to overlapping pockets of folded domains and Arg/Lys within IDRs; (3) ATP energy-independently induces protein folding with the highest efficiency known so far. As ATP likely emerged in the prebiotic monomeric world, while LLPS represents a pivotal mechanism to concentrate and compartmentalize rare molecules for forming primordial cells, ATP appears to control protein homeostasis and shape genome-proteome interfaces throughout the evolutionary trajectory, from prebiotic origins to modern cells.

Person-specific differences in ubiquitin-proteasome mediated proteostasis in human neurons.

BACKGROUND: Impairment of the ubiquitin-proteasome system (UPS) has been implicated in abnormal protein accumulation in Alzheimer's disease. It remains unclear if genetic variation affects the intrinsic properties of neurons that render some individuals more vulnerable to UPS impairment. METHODS: Induced pluripotent stem cell (iPSC)-derived neurons were generated from over 50 genetically variant and highly characterized participants of cohorts of aging. Proteomic profiling, proteasome activity assays, and Western blotting were employed to examine neurons at baseline and in response to UPS perturbation. RESULTS: Neurons with lower basal UPS activity were more vulnerable to tau accumulation following mild UPS inhibition. Chronic reduction in proteasome activity in human neurons induced compensatory elevation of regulatory proteins involved in proteostasis and several proteasome subunits. DISCUSSION: These findings reveal that genetic variation influences basal UPS activity in human neurons and differentially sensitizes them to external factors perturbing the UPS, leading to the accumulation of aggregation-prone proteins such as tau. HIGHLIGHTS: Polygenic risk score for AD is associated with the ubiquitin-proteasome system (UPS) in neurons. Basal proteasome activity correlates with aggregation-prone protein levels in neurons. Genetic variation affects the response to proteasome inhibition in neurons. Neuronal proteasome perturbation induces an elevation in specific proteins involved in proteostasis. Low basal proteasome activity leads to enhanced tau accumulation with UPS challenge.

The emerging roles of non-canonical ubiquitination in proteostasis and beyond.

Ubiquitin regulates various cellular functions by posttranslationally modifying substrates with diverse ubiquitin codes. Recent discoveries of new ubiquitin chain topologies, types of bonds, and non-protein substrates have substantially expanded the complexity of the ubiquitin code. Here, we describe the ubiquitin system covering the basic principles and recent discoveries related to mechanisms, technologies, and biological importance.

Fisetin alleviates cerebral ischemia/reperfusion injury by regulating Sirt1/Foxc1/Ubqln1 pathway-mediated proteostasis.

BACKGROUND: Cerebral ischemia/reperfusion injury (IRI) is pathologically associated with protein damage. The flavonoid fisetin has good therapeutic effects on cerebral IRI. However, the role of fisetin in regulating protein damage during cerebral IRI development remains unclear. This study investigated the pharmacological effects of fisetin on protein damage during cerebral IRI progression and defined the underlying mechanism of action. METHODS: In vivo and in vitro models of cerebral IRI were established by middle cerebral artery occlusion/reperfusion (MACO/R) and oxygen-glucose deprivation/reperfusion (OGD/R) treatment, respectively. Triphenyl tetrazolium chloride staining was performed to detect cerebral infarct size, and the modified neurologic severity score was used to examine neurological deficits. LDH activity and protein damage were assessed using kits. HT22 cell vitality and apoptosis were examined using CCK-8 assay and TUNEL staining, respectively. Interactions between Foxc1, Ubqln1, Sirt1, and Ezh2 were analyzed using CoIP, ChIP and/or dual-luciferase reporter gene assays. RESULTS: Fisetin alleviated protein damage and ubiquitinated protein aggregation and neuronal death caused by MCAO/R and OGD/R. Ubqln1 knockdown abrogated the inhibitory effect of fisetin on OGD/R-induced protein damage, ubiquitinated protein aggregation, and neuronal death in HT22 cells. Further experiments demonstrated that Foxc1 functions as a transcriptional activator of Ubqln1 and that Sirt1 promotes Foxc1 expression by deacetylating Ezh2 and inhibiting its activity. Furthermore, Sirt1 knockdown abrogated fisetin-mediated biological effects on OGD/R-treated HT22 cells. CONCLUSION: Fisetin improved proteostasis during cerebral IRI by regulating the Sirt1/Foxc1/Ubqln1 signaling axis. Our findings strongly suggest that fisetin-mediated inhibition of protein damage after ischemic stroke is a part of the mechanism through which fisetin is neuroprotective in cerebral IRI.

Mapping stress-responsive signaling pathways induced by mitochondrial proteostasis perturbations.

Imbalances in mitochondrial proteostasis are associated with pathologic mitochondrial dysfunction implicated in etiologically diverse diseases. This has led to considerable interest in defining the mechanisms responsible for regulating mitochondria in response to mitochondrial stress. Numerous stress-responsive signaling pathways have been suggested to regulate mitochondria in response to proteotoxic stress. These include the integrated stress response (ISR), the heat shock response (HSR), and the oxidative stress response (OSR). Here, we define the stress signaling pathways activated in response to chronic mitochondrial proteostasis perturbations by monitoring the expression of sets of genes regulated downstream of each of these signaling pathways in published Perturb-seq datasets from K562 cells CRISPRi-depleted of mitochondrial proteostasis factors. Interestingly, we find that the ISR is preferentially activated in response to chronic, genetically-induced mitochondrial proteostasis stress, with no other pathway showing significant activation. Further, we demonstrate that CRISPRi depletion of other mitochondria-localized proteins similarly shows preferential activation of the ISR relative to other stress-responsive signaling pathways. These results both establish our gene set profiling approach as a viable strategy to probe stress responsive signaling pathways induced by perturbations to specific organelles and identify the ISR as the predominant stress-responsive signaling pathway activated in response to chronic disruption of mitochondrial proteostasis.

Identification of molecular signatures defines the differential proteostasis response in induced spinal and cranial motor neurons.

Amyotrophic lateral sclerosis damages proteostasis, affecting spinal and upper motor neurons earlier than a subset of cranial motor neurons. To aid disease understanding, we exposed induced cranial and spinal motor neurons (iCrMNs and iSpMNs) to proteotoxic stress, under which iCrMNs showed superior survival, quantifying the transcriptome and proteome for >8,200 genes at 0, 12, and 36 h. Two-thirds of the proteome showed cell-type differences. iSpMN-enriched proteins related to DNA/RNA metabolism, and iCrMN-enriched proteins acted in the endoplasmic reticulum (ER)/ER chaperone complex, tRNA aminoacylation, mitochondria, and the plasma/synaptic membrane, suggesting that iCrMNs expressed higher levels of proteins supporting proteostasis and neuronal function. When investigating the increased proteasome levels in iCrMNs, we showed that the activity of the 26S proteasome, but not of the 20S proteasome, was higher in iCrMNs than in iSpMNs, even after a stress-induced decrease. We identified Ublcp1 as an iCrMN-specific regulator of the nuclear 26S activity.