Integrating Protein Homeostasis Strategies in Prokaryotes

Bacterial cells are frequently exposed to dramatic fluctuations in their environment, which cause perturbation in protein homeostasis and lead to protein misfolding. Bacteria have therefore evolved powerful quality control networks consisting of chaperones and proteases that cooperate to monitor the folding states of proteins and to remove misfolded conformers through either refolding or degradation. The levels of the quality control components are adjusted to the folding state of the cellular proteome through the induction of compartment specific stress responses. In addition, the activities of several quality control components are directly controlled by these stresses, allowing for fast activation. Severe stress can, however, overcome the protective function of the proteostasis network leading to the formation of protein aggregates, which are sequestered at the cell poles. Protein aggregates are either solubilized by AAA+ chaperones or eliminated through cell division, allowing for the generation of damage-free daughter cells.     

The fate of damaged mitochondrial DNA in the cell

Mitochondrion is a double membrane organelle that is responsible for cellular respiration and production of most of the ATP in eukaryotic cells. Mitochondrial DNA (mtDNA) is the genetic material carried by mitochondria, which encodes some essential subunits of respiratory complexes independent of nuclear DNA. Normally, mtDNA binds to certain proteins to form a nucleoid that is stable in mitochondria. Nevertheless, a variety of physiological or pathological stresses can cause mtDNA damage, and the accumulation of damaged mtDNA in mitochondria leads to mitochondrial dysfunction, which triggers the occurrence of mitochondrial diseases in vivo. In response to mtDNA damage, cell initiates multiple pathways including mtDNA repair, degradation, clearance and release, to recover mtDNA, and maintain mitochondrial quality and cell homeostasis. In this review, we provide our current understanding of the fate of damaged mtDNA, focus on the pathways and mechanisms of removing damaged mtDNA in the cell.     

Towards the control of intracellular protein turnover: mitochondrial Lon protease inhibitors versus proteasome inhibitors

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.     

Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis

Iron-sulfur (Fe-S) clusters are required for the functions of mitochondrial aconitase, mammalian iron regulatory protein 1, and many other proteins in multiple subcellular compartments. Recent studies in Saccharomyces cerevisiae indicated that Fe-S cluster biogenesis also has an important role in mitochondrial iron homeostasis. Here we report the functional analysis of the mitochondrial and cytosolic isoforms of the human Fe-S cluster scaffold protein, ISCU. Suppression of human ISCU by RNAi not only inactivated mitochondrial and cytosolic aconitases in a compartment-specific manner but also inappropriately activated the iron regulatory proteins and disrupted intracellular iron homeostasis. Furthermore, endogenous ISCU levels were suppressed by iron deprivation. These results provide evidence for a coordinated response to iron deficiency that includes activation of iron uptake, redistribution of intracellular iron, and decreased utilization of iron in Fe-S proteins.     

An overview of the role of molecular chaperones in protein homeostasis

Cells require a protein quality control (PQC) system to obtain a correct balance between folding and the degradation of incorrectly folded or misfolded proteins. This system maintains protein homeostasis and is essential for life. Key components of the PQC are molecular chaperones, which compose a ubiquitous class of proteins that mediate protein quality control by aiding in both the correct folding of proteins and the elimination of proteins that are misfolded due to cellular stress or mutation. Recent studies showed that protein homeostasis has an important role in nutrition and aging, increasing the relevance of the heat shock response to human health. This review summarizes our current knowledge of the molecular chaperone system and its role in protein homeostasis.     

Protein quality control at the plasma membrane

Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined.     

Ribosome and the secret of longevity

Proteins in cells are constantly undergoing damage. Nevertheless, the concentration of damaged proteins in young organisms is maintained at the low level due to the continual protein turnover: degradation of damaged proteins and synthesis of new ones. During aging, the concentration of damaged proteins increases because of decelerating protein turnover, the cause of which is unknown, however, it may be related to the decrease in ribosome concentration.     

Ribosome and the Secret of Longevity (A Hypothesis)

Proteins in cells are constantly undergoing damage. Nevertheless, the concentration of damaged proteins in young organisms is maintained at a low level owing to the continual protein turnover: breakdown of damaged proteins and synthesis of new ones. During aging, the concentration of damaged proteins increases because of decelerating protein turnover, the cause of which is unknown; however, it may be related to the decrease in ribosome concentration.     

The Immunoproteasome in oxidative stress,aging, and disease

The Immunoproteasome has traditionally been viewed primarily for its role in peptide production for antigen presentation by the major histocompatibility complex, which is critical for immunity. However, recent research has shown that the Immunoproteasome is also very important for the clearance of oxidatively damaged proteins in homeostasis, and especially during stress and disease. The importance of the Immunoproteasome in protein degradation has become more evident as diseases characterized by protein aggregates have also been linked to deficiencies of the Immunoproteasome. Additionally, there are now diseases defined by mutations or polymorphisms within Immunoproteasome-specific subunit genes, further suggesting its crucial role in cytokine signaling and protein homeostasis (or “proteostasis”). The purpose of this review is to highlight our growing understanding of the importance of the Immunoproteasome in the management of protein quality control, and the detrimental impact of its dysregulation during disease and aging.     

Oxidative protein damage and the proteasome

Protein damage, caused by radicals, is involved in many diseases and in the aging process. Therefore, it is crucial to understand how protein damage can be limited, repaired or removed. To degrade damaged proteins, several intracellular proteolytic systems exist. One of the most important contributors in intracellular protein degradation of oxidized, aggregated and misfolded proteins is the proteasomal system. The proteasome is not a simple, unregulated structure. It is a more complex proteolytic composition that undergoes diverse regulation in situations of oxidative stress, aging and pathology. In addition to that, numerous studies revealed that the proteasome activity is altered during life time, contributing to the aging process. In addition, in the nervous system, the proteasome plays an important role in maintaining neuronal protein homeostasis. However, alterations in the activity may have an impact on the onset of neurodegenerative diseases. In this review, we discuss what is presently known about protein damage, the role of the proteasome in the degradation of damaged proteins and how the proteasome is regulated. Special emphasis was laid on the role of the proteasome in neurodegenerative diseases.     

Unfolded protein responses in bacteria and mitochondria: a central role for the ClpXP machine

In the crowded environment of a cell, the protein quality control machinery, such as molecular chaperones and proteases, maintains a population of folded and hence functional proteins. The accumulation of unfolded proteins in a cell is particularly harmful as it not only reduces the concentration of active proteins but also overburdens the protein quality control machinery, which in turn, can lead to a significant increase in nonproductive folding and protein aggregation. To circumvent this problem, cells use heat shock and unfolded protein stress response pathways, which essentially sense the change to protein homeostasis upregulating protein quality control factors that act to restore the balance. Interestingly, several stress response pathways are proteolytically controlled. In this review, we provide a brief summary of targeted protein degradation by AAA+ proteases and focus on the role of ClpXP proteases, particularly in the signaling pathway of the Escherichia coli extracellular stress response and the mitochondrial unfolded protein response.     

Trehalose supplementation reduces hepatic endoplasmic reticulum stress and inflammatory signaling in old mice

The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging and cellular damage. Quality control systems, such as the unfolded protein response (UPR), inflammatory signaling and protein degradation, mitigate the residence time of damaged proteins. In the present study, we have examined the UPR and inflammatory signaling in the liver of young (~6 months) and old (~28 months) mice (n=8/group), and the ability of trehalose, a compound linked to increased protein stability and autophagy, to counteract age-induced effects on these systems. When used, trehalose was provided for 4 weeks in the drinking water immediately prior to sacrifice (n=7/group). Livers from old mice were characterized by activation of the UPR, increased inflammatory signaling and indices of liver injury. Trehalose treatment reduced the activation of the UPR and inflammatory signaling, and reduced liver injury. Reductions in proteins involved in autophagy and proteasome activity observed in old mice were restored following trehalose treatment. The autophagy marker, LC3B-II, was increased in old mice treated with trehalose. Metabolomics analyses demonstrated that reductions in hexosamine biosynthetic pathway metabolites and nicotinamide in old mice were restored following trehalose treatment. Trehalose appears to be an effective intervention to reduce age-associated liver injury and mitigate the need for activation of quality control systems that respond to disruption of proteostasis.     

Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein

Although minor abnormalities have been reported in prion protein (PrP) knock-out (Prnp-/-) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp-/- as compared to wild-type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin-protein conjugates were also observed in Prnp-/- mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.     

Tumor suppression by autophagy through the management of metabolic stress

Autophagy plays a critical protective role maintaining energy homeostasis and protein and organelle quality control. These functions are particularly important in times of metabolic stress and in cells with high energy demand such as cancer cells. In emerging cancer cells, autophagy defect may cause failure of energy homeostasis and protein and organelle quality control, leading to the accumulation of cellular damage in metabolic stress. Some manifestations of this damage, such as activation of the DNA damage response and generation of genome instability may promote tumor initiation and drive cell-autonomous tumor progression. In addition, in solid tumors, autophagy localizes to regions that are metabolically stressed. Defects in autophagy impair the survival of tumor cells in these areas, which is associated with increased cell death and inflammation. The cytokine response from inflammation may promote tumor growth and accelerate cell non-autonomous tumor progression. The overreaching theme is that autophagy protects cells from damage accumulation under conditions of metabolic stress allowing efficient tolerance and recovery from stress, and that this is a critical and novel tumor suppression mechanism. The challenge now is to define the precise aspects of autophagy, including energy homeostasis and protein and organelle turnover, that are required for the proper management of metabolic stress that suppress tumorigenesis. Furthermore, we need to be able to identify human tumors with deficient autophagy, and to develop rational cancer therapies that take advantage of the altered metabolic state and stress responses inherent to this autophagy defect.     

An unconventional pathway for mitochondrial protein degradation

Many vital metabolic pathways take place in mitochondria, but some of the associated processes generate toxic substances including reactive oxygen species that can damage proteins and DNA. Therefore, it is critical to maintain normally functioning mitochondria to achieve proper cellular homeostasis. Along these lines, mitochondrial dysfunction is associated with numerous diseases, and mitochondria quality control is essential for cell survival. The maintenance of functioning mitochondria is particularly important in aging cells, and there is a strong relationship between cellular aging and dysfunctional mitochondria. The best characterized pathway that is responsible for the elimination of damaged mitochondria is mitophagy, a selective type of autophagy. In yeast, mitophagy requires the mitochondrial protein Atg32 to serve as a receptor for recognition and sequestration by a phagophore. Although conventional mitophagy has been extensively studied, recent research suggests that an unconventional pathway, which is independent of Atg32, contributes to the removal of mitochondria.     

Restricted access: spatial sequestration of damaged proteins during stress and aging

The accumulation of damaged and aggregated proteins is a hallmark of aging and increased proteotoxic stress. To limit the toxicity of damaged and aggregated proteins and to ensure that the damage is not inherited by succeeding cell generations, a system of spatial quality control operates to sequester damaged/aggregated proteins into inclusions at specific protective sites. Such spatial sequestration and asymmetric segregation of damaged proteins have emerged as key processes required for cellular rejuvenation. In this review, we summarize findings on the nature of the different quality control sites identified in yeast, on genetic determinants required for spatial quality control, and on how aggregates are recognized depending on the stress generating them. We also briefly compare the yeast system to spatial quality control in other organisms. The data accumulated demonstrate that spatial quality control involves factors beyond the canonical quality control factors, such as chaperones and proteases, and opens up new venues in approaching how proteotoxicity might be mitigated, or delayed, upon aging.     

CHIP deficiency decreases longevity, with accelerated aging phenotypes accompanied by altered protein quality control

During the course of biological aging, there is a gradual accumulation of damaged proteins and a concomitant functional decline in the protein degradation system. Protein quality control is normally ensured by the coordinated actions of molecular chaperones and the protein degradation system that collectively help to maintain protein homeostasis. The carboxyl terminus of Hsp70-interacting protein (CHIP), a ubiquitin ligase/cochaperone, participates in protein quality control by targeting a broad range of chaperone substrates for proteasome degradation via the ubiquitin-proteasome system, demonstrating a broad involvement of CHIP in maintaining cytoplasmic protein quality control. In the present study, we have investigated the influence that protein quality control exerts on the aging process by using CHIP-/- mice. CHIP deficiency in mice leads to a markedly reduced life span, along with accelerated age-related pathophysiological phenotypes. These features were accompanied by indications of accelerated cellular senescence and increased indices of oxidative stress. In addition, CHIP-/- mice exhibit a deregulation of protein quality control, as indicated by elevated levels of toxic oligomer proteins and a decline in proteasome activity. Taken together, these data reveal that impaired protein quality control contributes to cellular senescence and implicates CHIP-dependent quality control mechanisms in the regulation of mammalian longevity in vivo.     

Mitochondria drive autophagy pathology via microtubule disassembly

Neurons are exquisitely dependent on quality control systems to maintain a healthy intracellular environment. A permanent assessment of protein and organelle “quality” allows a coordinated action between repair and clearance of damage proteins and dysfunctional organelles. Impairments in the intracellular clearance mechanisms in long-lived postmitotic cells, like neurons, result in the progressive accumulation of damaged organelles and aggregates of aberrant proteins. Using cells bearing Parkinson disease (PD) patients’ mitochondria, we demonstrated that aberrant accumulation of autophagosomes in PD, commonly interpreted as an abnormal induction of autophagy, is instead due to defective autophagic clearance. This defect is a consequence of alterations in the microtubule network driven by mitochondrial dysfunction that hinder mitochondria and autophagosome trafficking. We uncover mitochondria and microtubule-directed traffic as main players in the regulation of autophagy in PD.     

Tumor suppression by autophagy through the management of metabolic stress

Autophagy plays a critical protective role maintaining energy homeostasis and protein and organelle quality control. These functions are particularly important in times of metabolic stress and in cells with high energy demand such as cancer cells. In emerging cancer cells, autophagy defect may cause failure of energy homeostasis and protein and organelle quality control, leading to the accumulation of cellular damage in metabolic stress. Some manifestations of this damage, such as activation of the DNA damage response and generation of genome instability may promote tumor initiation and drive cell-autonomous tumor progression. In addition, in solid tumors, autophagy localizes to regions that are metabolically stressed. Defects in autophagy impair the survival of tumor cells in these areas, which is associated with increased cell death and inflammation. The cytokine response from inflammation may promote tumor growth and accelerate cell non-autonomous tumor progression. The overreaching theme is that autophagy protects cells from damage accumulation under conditions of metabolic stress allowing efficient tolerance and recovery from stress, and that this is a critical and novel tumor suppression mechanism. The challenge now is to define the precise aspects of autophagy, including energy homeostasis, and protein and organelle turnover, that are required for the proper management of metabolic stress that suppress tumorigenesis. Furthermore, we need to be able to identify human tumors with deficient autophagy, and to develop rational cancer therapies that take advantage of the altered metabolic state and stress responses inherent to this autophagy defect.