Protein Quality Control System Research Articles

How and why do toxic conformers of aberrant proteins accumulate during ageing?

Ageing can be defined as a gradual decline in cellular and physical functions accompanied by an increased sensitivity to the environment and risk of death. The increased risk of mortality is causally connected to a gradual, intracellular accumulation of so-called ageing factors, of which damaged and aggregated proteins are believed to be one. Such aggregated proteins also contribute to several age-related neurodegenerative disorders e.g. Alzheimer's, Parkinson's, and Huntington's diseases, highlighting the importance of protein quality control (PQC) in ageing and its associated diseases. PQC consists of two interrelated systems: the temporal control system aimed at refolding, repairing, and/or removing aberrant proteins and their aggregates and the spatial control system aimed at harnessing the potential toxicity of aberrant proteins by sequestering them at specific cellular locations. The accumulation of toxic conformers of aberrant proteins during ageing is often declared to be a consequence of an incapacitated temporal PQC system-i.e. a gradual decline in the activity of chaperones and proteases. Here, we review the current knowledge on PQC in relation to ageing and highlight that the breakdown of both temporal and spatial PQC may contribute to ageing and thus comprise potential targets for therapeutic interventions of the ageing process.

E3 Ubiquitin Ligases Neurobiological Mechanisms: Development to Degeneration.

Cells regularly synthesize new proteins to replace old or damaged proteins. Deposition of various aberrant proteins in specific brain regions leads to neurodegeneration and aging. The cellular protein quality control system develop various defense mechanisms against the accumulation of misfolded and aggregated proteins. The mechanisms underlying the selective recognition of specific crucial protein or misfolded proteins are majorly governed by quality control E3 ubiquitin ligases mediated through ubiquitin-proteasome system. Few known E3 ubiquitin ligases have shown prominent neurodevelopmental functions, but their interactions with different developmental proteins play critical roles in neurodevelopmental disorders. Several questions are yet to be understood properly. How E3 ubiquitin ligases determine the specificity and regulate degradation of a particular substrate involved in neuronal proliferation and differentiation is certainly the one, which needs detailed investigations. Another important question is how neurodevelopmental E3 ubiquitin ligases specifically differentiate between their versatile range of substrates and timing of their functional modulations during different phases of development. The premise of this article is to understand how few E3 ubiquitin ligases sense major molecular events, which are crucial for human brain development from its early embryonic stages to throughout adolescence period. A better understanding of these few E3 ubiquitin ligases and their interactions with other potential proteins will provide invaluable insight into disease mechanisms to approach toward therapeutic interventions.

To CURe or not to CURe? Differential effects of the chaperone sorting factor Cur1 on yeast prions are mediated by the chaperone Sis1.

Yeast self-perpetuating protein aggregates (prions) provide a convenient model for studying various components of the cellular protein quality control system. Molecular chaperones and chaperone-sorting factors, such as yeast Cur1 protein, play key role in proteostasis via tight control of partitioning and recycling of misfolded proteins. In this study, we show that, despite the previously described ability of Cur1 to antagonize the yeast prion [URE3], it enhances propagation and phenotypic manifestation of another prion, [PSI+ ]. We demonstrate that both curing of [URE3] and enhancement of [PSI+ ] in the presence of excess Cur1 are counteracted by the cochaperone Hsp40-Sis1 in a dosage-dependent manner, and show that the effect of Cur1 on prions parallels effects of the attachment of nuclear localization signal to Sis1, indicating that Cur1 acts on prions via its previously reported ability to relocalize Sis1 from the cytoplasm to nucleus. This shows that the direction in which Cur1 influences a prion depends on how this specific prion responds to relocalization of Sis1.

Molecular Basis of Human Diseases and Targeted Therapy Based on Small-Molecule Inhibitors of ER Stress-Induced Signaling Pathways.

The Endoplasmic Reticulum (ER) provides a conserved protein quality control system and plays a fundamental role in cell growth and homeostasis. Disturbances in the ER homeostasis may originate especially from hypoxia, glucose deficiency, presence of mutant proteins, that directly impair protein folding capacity and after deposition of unfolded and misfolded proteins within ER lumen trigger ER stress conditions. This subsequently activates the Unfolded Protein Response (UPR) branches, which have a dual pro-adaptive or pro-apoptotic role depending on the severity and time of duration of ER stress conditions. This review is the first to offer a detailed overview on molecular mechanisms of all major ER stress-dependent signaling branches, that are activated through three specific ER transmembrane receptors of impaired protein folding: Protein kinase RNA (PKR)-like ER kinase (PERK), Inositol-requiring enzyme-1 (IRE1) and Activating transcription factor 6 (ATF6). Molecular crosstalk among ER transmembrane receptors-dependent pathways determines a final UPR response, but the recent data reported that especially PERK over-activation has a significant impact on the development and progression of a wide spectrum of disease entities. Based on these findings, small-molecules, highly specific PERK inhibitors may provide effective, groundbreaking treatment strategy against human diseases. However, after foregoing in vitro cellular and in vivo animal models conducted examination, supplementary investigations of PERK inhibitors are required for their further clinical use. Future research may answer the question of how to minimize toxicity and side effects of characterized small-molecule PERK inhibitors, that may be used, as breakthrough drugs, alone or in combination with currently known models of therapy.

Stress induced nuclear granules form in response to accumulation of misfolded proteins in Caenorhabditis elegans

BackgroundEnvironmental stress can affect the viability or fecundity of an organism. Environmental stressors may affect the genome or the proteome and can cause cellular distress by contributing to protein damage or misfolding. This study examines the cellular response to environmental stress in the germline of the nematode, C. elegans.ResultsSalt stress, oxidative stress, and starvation, but not heat shock, induce the relocalization of ubiquitin, proteasome, and the TIAR-2 protein into distinct subnuclear regions referred to as stress induced nuclear granules (SINGs). The SINGs form within 1 h of stress initiation and do not require intertissue signaling. K48-linked polyubiquitin chains but not K63 chains are enriched in SINGs. Worms with a mutation in the conjugating enzyme, ubc-18, do not form SINGs. Additionally, knockdown of ubc-20 and ubc-22 reduces the level of SING formation as does knockdown of the ubiquitin ligase chn-1, a CHIP homolog. The nuclear import machinery is required for SING formation. Stressed embryos containing SINGs fail to hatch and cell division in these embryos is halted. The formation of SINGs can be prevented by pre-exposure to a brief period of heat shock before stress exposure. Heat shock inhibition of SINGs is dependent upon the HSF-1 transcription factor.ConclusionsThe heat shock results suggest that chaperone expression can prevent SING formation and that the accumulation of damaged or misfolded proteins is a necessary precursor to SING formation. Thus, SINGs may be part of a novel protein quality control system. The data suggest an interesting model where SINGs represent sites of localized protein degradation for nuclear or cytosolic proteins. Thus, the physiological impacts of environmental stress may begin at the cellular level with the formation of stress induced nuclear granules.

Dissection of ubiquitinated protein degradation by basal autophagy.

Basal autophagy plays an essential role as a protein quality control system. Although it has been demonstrated that the loss of autophagy results in the accumulation of ubiquitin‐positive aggregates and the development of neurodegenerative diseases, the precise autophagy substrate(s) remain unclear. Here, we determined whether ubiquitinated proteins are direct substrates for basal autophagy using a fluorescent ratiometric probe for ubiquitin. We show that the degradation of polyubiquitinated proteins is not dependent on basal autophagy. Although ubiquitin‐positive aggregates are observed in autophagy knockout cultured cells, the aggregates consist of soluble and mobile polyubiquitinated proteins, which are trapped by p62 without an increase in the total amount of ubiquitinated proteins. These results suggest that ubiquitinated proteins are not major targets for basal autophagy.

The Arabidopsis endoplasmic reticulum associated degradation pathways are involved in the regulation of heat stress response

The Cytosolic Protein Response (CPR) in the cytosol and the Unfolded Protein Response (UPR) and ER-associated degradation (ERAD) in the endoplasmic reticulum are major pathways of the cellular proteostasis network. However, despite years of effort, how these protein quality control systems coordinated invivo remains largely unknown, particularly in plants. In this study, the roles of two evolutionarily conserved ERAD pathways (DOA10 and HRD1) in heat stress response were investigated through reverse genetic approaches in Arabidopsis. Phenotypic analysis of the mutants showed that the two ERAD pathways additively play negative roles in heat tolerance, which was demonstrated by higher survival rate and lower electrolyte leakage in the loss of function mutants compared to the wild type plants. Importantly, gene expression analysis revealed that the mutant plants showed elevated transcriptional regulation of several downstream genes, including those encoding CPR and UPR marker genes, under both basal and heat stress conditions. Finally, multiple components of ERAD genes exhibited rapid response to increasing temperature. Taken together, our data not only unravels key insights into the crosstalk between different protein quality control processes, but also provides candidate genes to genetically improve plant heat tolerance in the future.

Transmembrane domain quality control systems operate at the endoplasmic reticulum and Golgi apparatus.

Multiple protein quality control systems operate to ensure that misfolded proteins are efficiently cleared from the cell. While quality control systems that assess the folding status of soluble domains have been extensively studied, transmembrane domain (TMD) quality control mechanisms are poorly understood. Here, we have used chimeras based on the type I plasma membrane protein CD8 in which the endogenous TMD was substituted with transmembrane sequences derived from different polytopic membrane proteins as a mode to investigate the quality control of unassembled TMDs along the secretory pathway. We find that the three TMDs examined prevent trafficking of CD8 to the cell surface via potentially distinct mechanisms. CD8 containing two distinct non-native transmembrane sequences escape the ER and are subsequently retrieved from the Golgi, possibly via Rer1, leading to ER localisation at steady state. A third chimera, containing an altered transmembrane domain, was predominantly localised to the Golgi at steady state, indicating the existence of an additional quality control checkpoint that identifies non-native transmembrane domains that have escaped ER retention and retrieval. Preliminary experiments indicate that protein retained by quality control mechanisms at the Golgi are targeted to lysosomes for degradation.

Decreased Levels of Foldase and Chaperone Proteins Are Associated with an Early-Onset Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive upper and lower motor neuron degeneration. One of the peculiar clinical characteristics of ALS is the wide distribution in age of onset, which is probably caused by different combinations of intrinsic and exogenous factors. We investigated whether these modifying factors are converging into common pathogenic pathways leading either to an early or a late disease onset. This would imply the identification of phenotypic biomarkers, that can distinguish the two populations of ALS patients, and of relevant pathways to consider in a therapeutic intervention. Toward this aim a differential proteomic analysis was performed in peripheral blood mononuclear cells (PBMC) from a group of 16 ALS patients with an age of onset ≤55 years and a group of 16 ALS patients with an age of onset ≥75 years, and matched healthy controls. We identified 43 differentially expressed proteins in the two groups of patients. Gene ontology analysis revealed that there was a significant enrichment in annotations associated with protein folding and response to stress. We next validated a selected number of proteins belonging to this functional group in 85 patients and 83 age- and sex-matched healthy controls using immunoassays. The results of the validation study confirmed that there was a decreased level of peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin A), heat shock protein HSP 90-alpha, 78 kDa glucose-regulated protein (also known as BiP) and protein deglycase DJ-1 in PBMC of ALS patients with an early onset. Similar results were obtained in PBMC and spinal cord from two SOD1G93A mouse models with an early and late disease onset. This study suggests that a different ability to upregulate proteins involved in proteostasis, such as foldase and chaperone proteins, may be at the basis of a different susceptibility to ALS, putting forward the development of therapeutic approaches aiming at boosting the protein quality control system.

The growing world of small heat shock proteins: from structure to functions.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation.

ABSTRACTMultiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation.

The Involvement of Heat Shock Proteins in the Establishment of Tomato Yellow Leaf Curl Virus Infection.

Tomato yellow leaf curl virus (TYLCV), a begomovirus, induces protein aggregation in infected tomatoes and in its whitefly vector Bemisia tabaci. The interactions between TYLCV and HSP70 and HSP90 in plants and vectors are necessity for virus infection to proceed. In infected host cells, HSP70 and HSP90 are redistributed from a soluble to an aggregated state. These aggregates contain, together with viral DNA/proteins and virions, HSPs and components of the protein quality control system such as ubiquitin, 26S proteasome subunits, and the autophagy protein ATG8. TYLCV CP can form complexes with HSPs in tomato and whitefly. Nonetheless, HSP70 and HSP90 play different roles in the viral cell cycle in the plant host. In the infected host cell, HSP70, but not HSP90, participates in the translocation of CP from the cytoplasm into the nucleus. Viral amounts decrease when HSP70 is inhibited, but increase when HSP90 is downregulated. In the whitefly vector, HSP70 impairs the circulative transmission of TYLCV; its inhibition increases transmission. Hence, the efficiency of virus acquisition by whiteflies depends on the functionality of both plant chaperones and their cross-talk with other protein mechanisms controlling virus-induced aggregation.

A critical role of Hrd1 in the regulation of optineurin degradation and aggresome formation.

Mutations in optineurin (OPTN) are associated with several human disorders including amyotrophic lateral sclerosis (ALS) and primary open-angle glaucoma (POAG). OPTN is known to be a multifunctional autophagy receptor that plays important roles in NF-κB signaling, vesicle trafficking, maintenance of the Golgi apparatus and autophagy. Given that a loss of neurons and an abnormal aggregation of disease proteins are two key features of neurodegenerative diseases, protein quality control systems are considered to be tightly associated with neurodegeneration. In this study, we investigated the involvement of the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway, two major intracellular protein quality control systems, in the regulation of wild-type (WT) OPTN, ALS-linked mutant E478G OPTN and POAG-linked mutant E50K OPTN. Our data revealed that the UPS, not the autophagy-lysosome pathway, is the major system for degradation and aggregation of OPTN. Moreover, we found that Hrd1, an E3 ubiquitin ligase, could play an important role in the protein quality control of OPTN. Our results demonstrated that overexpression of Hrd1 increased the proteasomal degradation and microtubule-dependent aggresome formation of OPTN in the microtubular organizing center, whereas knockdown of Hrd1 stabilized OPTN and inhibited aggresome formation of OPTN.

Noncoding RNAs in protein clearance pathways: implications in neurodegenerative diseases.

The importance of noncoding genome has become more evident in recent years. Before genome sequencing, the most well studied portion of our genome was protein coding genes. Interestingly, this coding portion accounted only for 1.5% of the genome, the rest being the noncoding sequences. Noncoding RNAs (ncRNAs) are involved in normal cell physiology, stress, and disease states. A class of small ncRNAs and miRNAs has gained much importance because of its involvement in human diseases such as cancer. Involvement of long ncRNAs have also been acknowledged in other human diseases, especially in neurodegenerative diseases. Neurodegenerative diseases are characterized by the presence of abnormally folded proteins that are toxic to the cell. Several studies from model organisms suggest upregulation of pathways that clear this toxic protein may provide protection against neurodegeneration. In this review, I summarize the importance of ncRNAs in protein quality control system of cell that is implicated in this fatal group of neurodegenerative diseases.

Cellular Regulation of Amyloid Formation in Aging and Disease.

As the population is aging, the incidence of age-related neurodegenerative diseases, such as Alzheimer and Parkinson disease, is growing. The pathology of neurodegenerative diseases is characterized by the presence of protein aggregates of disease specific proteins in the brain of patients. Under certain conditions these disease proteins can undergo structural rearrangements resulting in misfolded proteins that can lead to the formation of aggregates with a fibrillar amyloid-like structure. Cells have different mechanisms to deal with this protein aggregation, where the molecular chaperone machinery constitutes the first line of defense against misfolded proteins. Proteins that cannot be refolded are subjected to degradation and compartmentalization processes. Amyloid formation has traditionally been described as responsible for the proteotoxicity associated with different neurodegenerative disorders. Several mechanisms have been suggested to explain such toxicity, including the sequestration of key proteins and the overload of the protein quality control system. Here, we review different aspects of the involvement of amyloid-forming proteins in disease, mechanisms of toxicity, structural features, and biological functions of amyloids, as well as the cellular mechanisms that modulate and regulate protein aggregation, including the presence of enhancers and suppressors of aggregation, and how aging impacts the functioning of these mechanisms, with special attention to the molecular chaperones.

Lipid profiling of parkin-mutant human skin fibroblasts.

Parkin mutations are a major cause of early-onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin-mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) and thin-layer chromatography (TLC). In parallel, we have performed direct MALDI-TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin-mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well-known component of PD.

Role of sHsps in organizing cytosolic protein aggregation and disaggregation.

Small heat shock proteins (sHsps) exhibit an ATP-independent chaperone activity to prevent the aggregation of misfolded proteins in vitro. The seemingly conflicting presence of sHsps in insoluble protein aggregates in cells obstructs a precise definition of sHsp function in proteostasis networks. Recent findings specify sHsp activities in protein quality control systems. The sHsps of yeast, Hsp42 and Hsp26, interact with early unfolding intermediates of substrates, keeping them in a ready-to-refold conformation close to the native state. This activity facilitates substrate refolding by ATP-dependent Hsp70-Hsp100 disaggregating chaperones. Hsp42 can actively sequester misfolded proteins and promote their deposition at specific cellular sites. This aggregase activity represents a cytoprotective protein quality control strategy. The aggregase function of Hsp42 controls the formation of cytosolic aggregates (CytoQs) under diverse stress regimes and can be reconstituted in vitro, demonstrating that Hsp42 is necessary and sufficient to promote protein aggregation. Substrates sequestered at CytoQs can be dissociated by Hsp70-Hsp100 disaggregases for subsequent triage between refolding and degradation pathways or are targeted for destruction by selective autophagy termed proteophagy.

Loss of Sigma-1 Receptor Chaperone Promotes Astrocytosis and Enhances the Nrf2 Antioxidant Defense.

Sigma-1 receptor (Sig-1R) functions as a chaperon that interacts with multiple proteins and lipids and is implicated in neurodegenerative and psychiatric diseases. Here, we used Sig-1R KO mice to examine brain expression profiles of astrocytes and ubiquitinated proteins, which are both hallmarks of central nervous system (CNS) pathologies. Our results showed that Sig-1R KO induces increased glial fibrillary acidic protein (GFAP) expression in primary neuron-glia cultures and in the whole brain of fetus mice with concomitantly increased accumulations of ubiquitinated proteins. Astrogliosis was also observed in the neuron-glia culture. Upon proteasome or autophagy inhibitor treatments, the pronounced ubiquitinated proteins were further increased in Sig-1R KO neurons, indicating that the Sig-1R regulates both protein degradation and quality control systems. We found that Nrf2 (nuclear factor erythroid 2-related factor 2), which functions to overcome the stress condition, was enhanced in the Sig-1R KO systems especially when cells were under stressful conditions. Mutation or deficiency of Sig-1Rs has been observed in neurodegenerative models. Our study identifies the critical roles of Sig-1R in CNS homeostasis and supports the idea that functional complementation pathways are triggered in the Sig-1R KO pathology.

Resveratrol Attenuates Copper-Induced Senescence by Improving Cellular Proteostasis.

Copper sulfate-induced premature senescence (CuSO4-SIPS) consistently mimetized molecular mechanisms of replicative senescence, particularly at the endoplasmic reticulum proteostasis level. In fact, disruption of protein homeostasis has been associated to age-related cell/tissue dysfunction and human disorders susceptibility. Resveratrol is a polyphenolic compound with proved antiaging properties under particular conditions. In this setting, we aimed to evaluate resveratrol ability to attenuate cellular senescence induction and to unravel related molecular mechanisms. Using CuSO4-SIPS WI-38 fibroblasts, resveratrol is shown to attenuate typical senescence alterations on cell morphology, senescence-associated beta-galactosidase activity, and cell proliferation. The mechanisms implicated in this antisenescence effect seem to be independent of senescence-associated genes and proteins regulation but are reliant on cellular proteostasis improvement. In fact, resveratrol supplementation restores copper-induced increased protein content, attenuates BiP level, and reduces carbonylated and polyubiquitinated proteins by autophagy induction. Our data provide compelling evidence for the beneficial effects of resveratrol by mitigating CuSO4-SIPS stressful consequences by the modulation of protein quality control systems. These findings highlight the importance of a balanced cellular proteostasis and add further knowledge on molecular mechanisms mediating resveratrol antisenescence effects. Moreover, they contribute to identifying specific molecular targets whose modulation will prevent age-associated cell dysfunction and improve human healthspan.

THU-398 - Induced pluripotent stem cell-derived hepatocyte-like cells for study of the TTR protein quality control system

THU-398 - Induced pluripotent stem cell-derived hepatocyte-like cells for study of the TTR protein quality control system