Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain

In order to successfully respond to stress all cells rely on the ability of the proteasomal and lysosomal proteolytic pathways to continually maintain protein turnover. Increasing evidence suggests that as part of normal aging there are age-related impairments in protein turnover by the proteasomal proteolytic pathway, and perturbations of the lysosomal proteolytic pathway. Furthermore, with numerous studies suggest an elevated level of a specialized form of lysosomal proteolysis (autophagy or macroautophagy) occurs during the aging of multiple cell types. Age-related alterations in proteolysis are believed to contribute to a wide variety of neuropathological manifestations including elevations in protein oxidation, protein aggregation, and cytotoxicity. Within the brain altered protein turnover is believed to contribute to elevations in multiple forms of protein aggregation ranging from tangle and Lewy body formation, to lipofuscin-ceroid accumulation. In this review we discuss and summarize evidence for proteolytic alterations occurring in the aging brain, the contribution of oxidative stress to disruption of protein turnover during normal aging, the evidence for cross-talk between the proteasome and lysosomal proteolytic pathways in the brain, and explore the contribution of altered proteolysis as a mediator of oxidative stress, neuropathology, and neurotoxicity in the aging brain.     

Protein homeostasis and aging: role of ubiquitin protein ligases

Protein homeostasis is fundamental in normal cellular function and cell survival. The ubiquitin-proteasome system (UPS) plays a central role in maintaining the protein homeostasis network through selective elimination of misfolded and damaged proteins. Impaired function of UPS is implicated in normal aging process and also in several age-related neurodegenerative disorders that are characterized by increased accumulation oxidatively modified proteins and protein aggregates. Growing literature also indicate the potential role of various ubiquitin protein ligases in the regulation of aging process by enhancing the degradation of either central lifespan regulators or abnormally folded and damaged proteins. This review mainly focuses on our current understanding of the importance of UPS function in the regulation of normal aging process.     

Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.     

SKN-1/Nrf,stress responses,and aging in Caenorhabditis elegans

The mammalian Nrf/CNC proteins (Nrf1, Nrf2, Nrf3, p45 NF-E2) perform a wide range of cellular protective and maintenance functions. The most thoroughly described of these proteins, Nrf2, is best known as a regulator of antioxidant and xenobiotic defense, but more recently has been implicated in additional functions that include proteostasis and metabolic regulation. In the nematode Caenorhabditis elegans, which offers many advantages for genetic analyses, the Nrf/CNC proteins are represented by their ortholog SKN-1. Although SKN-1 has diverged in aspects of how it binds DNA, it exhibits remarkable functional conservation with Nrf/CNC proteins in other species and regulates many of the same target gene families. C. elegans may therefore have considerable predictive value as a discovery model for understanding how mammalian Nrf/CNC proteins function and are regulated in vivo. Work in C. elegans indicates that SKN-1 regulation is surprisingly complex and is influenced by numerous growth, nutrient, and metabolic signals. SKN-1 is also involved in a wide range of homeostatic functions that extend well beyond the canonical Nrf2 function in responses to acute stress. Importantly, SKN-1 plays a central role in diverse genetic and pharmacologic interventions that promote C. elegans longevity, suggesting that mechanisms regulated by SKN-1 may be of conserved importance in aging. These C. elegans studies predict that mammalian Nrf/CNC protein functions and regulation may be similarly complex and that the proteins and processes that they regulate are likely to have a major influence on mammalian life- and healthspan.     

Peptides that activate the 20S proteasome by gate opening increased oxidized protein removal and reduced protein aggregation

The proteasome is a multicatalytic protease that is responsible for the degradation of the majority of intracellular proteins. Its role is correlated with several major regulatory pathways that are involved in cell cycle control, signaling, and antigen presentation, as well as in the removal of oxidatively damaged proteins. Although several proteasomal catalytic inhibitors have been described, very few activators have been reported to date. Some reports in the literature highlight the cellular protective effects of proteasome activation against oxidative stress and its effect on increased life span. In this work, we describe a peptide named proteasome-activating peptide 1 (PAP1), which increases the chymotrypsin-like proteasomal catalytic activity and, consequently, proteolytic rates both in vitro and in culture. PAP1 proteasomal activation is mediated by the opening of the proteasomal catalytic chamber. We also demonstrate that the observed proteasomal activation protected cells from oxidative stress; further, PAP1 prevented protein aggregation in a cellular model of amyotrophic lateral sclerosis. The role of 20SPT gate opening underlying protection against oxidative stress was also explored in yeast cells. The present data indicate the importance of proteasomal activators as potential drugs for the treatment of pathologies associated with the impaired removal of damaged proteins, which is observed in many neurodegenerative diseases.     

Identification and characterization of protein cross-links induced by oxidative reactions

Introduction: Protein cross-links are common in biological systems and are generated both deliberately as a part of normal metabolism and also accidently as a result of exposure to external factors such as oxidation and glycation stresses. These cross-links can be both positive and negative for biological function, with high levels of inappropriate cross-links being associated with multiple human pathologies, as well as loss of protein structure and function in the food, agricultural, and pharmaceutical fields.

Areas covered: This review covers recent advances in the detection and identification of protein cross-links arising from oxidation reactions, mediated by both radicals and two-electron oxidants. Information on both enzymatic and nonenzymatic cross-linking is reviewed, both where this is intentional, as part of normal metabolism, and accidental and a potential cause of disease.

Expert commentary: The advantages and drawbacks of various methods available for the detection, identification, and quantification of these species are discussed, as well as some of the mechanisms and processes known to give rise to these species.     

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.     

Proteasomal dysfunction and endoplasmic reticulum stress enhance trafficking of prion protein aggregates through the secretory pathway and increase accumulation of pathologic prion protein

A conformational change of the cellular prion protein (PrP(c)) underlies formation of PrP(Sc), which is closely associated with pathogenesis and transmission of prion diseases. The precise conformational prerequisites and the cellular environment necessary for this post-translational process remain to be completely elucidated. At steady state, glycosylated PrP(c) is found primarily at the cell surface, whereas a minor fraction of the population is disposed of by the ER-associated degradation-proteasome pathway. However, chronic ER stress conditions and proteasomal dysfunctions lead to accumulation of aggregation-prone PrP molecules in the cytosol and to neurodegeneration. In this study, we challenged different cell lines by inducing ER stress or inhibiting proteasomal activity and analyzed the subsequent repercussion on PrP metabolism, focusing on PrP in the secretory pathway. Both events led to enhanced detection of PrP aggregates and a significant increase of PrP(Sc) in persistently prion-infected cells, which could be reversed by overexpression of proteins of the cellular quality control. Remarkably, upon proteasomal impairment, an increased fraction of misfolded, fully glycosylated PrP molecules traveled through the secretory pathway and reached the plasma membrane. These findings suggest a novel pathway that possibly provides additional substrate and template necessary for prion formation when protein clearance by the proteasome is impaired.     

Proteasome alterations during adipose differentiation and aging: links to impaired adipocyte differentiation and development of oxidative stress

Intracellular proteins are degraded by a number of proteases, including the ubiquitin-proteasome pathway (UPP). Impairments in the UPP occur during the aging of a variety of tissues, although little is known in regards to age-related alterations to the UPP during the aging of adipose tissue. The UPP is known to be involved in regulating the differentiation of a variety of cell types, although the potential changes in the UPP during adipose differentiation have not been fully elucidated. How the UPP is altered in aging adipose tissue and adipocyte differentiation and the effects of proteasome inhibition on adipocyte homeostasis and differentiation are critical issues to elucidate experimentally. Adipogenesis continues throughout the life of adipose tissue, with continual differentiation of preadipocytes essential to maintaining tissue function during aging, and UPP alterations in mature adipocytes are likely to directly modulate adipose function during aging. In this study we demonstrate that aging induces alterations in the activity and expression of principal components of the UPP. Additionally, we show that multiple changes in the UPP occur during the differentiation of 3T3-L1 cells into adipocytes. In vitro data link observed UPP alterations to increased levels of oxidative stress and altered adipose biology relevant to both aging and differentiation. Taken together, these data demonstrate that changes in the UPP occur in response to adipose aging and adipogenesis and strongly suggest that proteasome inhibition is sufficient to decrease adipose differentiation, as well as increasing oxidative stress in mature adipocytes, both of which probably promote deleterious effects on adipose aging.     

The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver

The circadian clock regulates many cellular processes, notably including the cell cycle, metabolism and aging. Mitochondria play essential roles in metabolism and are the major sites of reactive oxygen species (ROS) production in the cell. The clock regulates mitochondrial functions by driving daily changes in NAD⁺ levels and Sirt3 activity. In addition to this central route, in the present study, we find that the expression of some mitochondrial genes is also rhythmic in the liver, and that there rhythms are disrupted by the Clock^Δ19 mutation in young mice, suggesting that they are regulated by the core circadian oscillator. Related to this observation, we also find that the regulation of oxidative stress is rhythmic in the liver. Since mitochondria and ROS play important roles in aging, and mitochondrial functions are also disturbed by aging, these related observations prompt the compelling hypothesis that circadian oscillators influence aging by regulating ROS in mitochondria. During aging, the expression rhythms of some mitochondrial genes were altered in the liver and the temporal regulation over the dynamics of mitochondrial oxidative stress was disrupted. However, the expression of clock genes was not affected. Our results suggested that mitochondrial functions are combinatorially regulated by the clock and other age-dependent mechanism(s), and that aging disrupts mitochondrial rhythms through mechanisms downstream of the clock.     

Photosensitized reactions mediated by the major chromophore arising from glucose decomposition, result in oxidation and cross-linking of lens proteins and activation of the proteasome

Glucose solutions incubated at low oxygen concentration gave rise to the appearance of an absorption band in the UVA-visible region after 10 days. Further characterization evidenced that this band was composed by a single chomophore with maximum absorption bands at 335 and 365 nm. HPLC/MS and UV spectroscopy assays indicated that this product is composed by five unities of furan. Importantly, the presence of a compound with identical spectral and chromatographic properties was observed in the water-soluble fraction of cataractous human eye lenses. The photo-biological effects of this glucose-derived chromophore (GDC) have been addressed using targets of biological relevance, such as water-soluble proteins from eye lens and the proteasome present in this protein mixture. Increased protein oxidation and protein crosslinking was observed when lens proteins were exposed to UVA-visible light in the presence of GDC under a 5% and 20% oxygen atmosphere. In addition, an increased proteasome peptidase activity was also observed. However, the use of D₂O resulted in decreased proteasome activity, suggesting that singlet oxygen promotes the impairment of proteasome activity. Our results suggest that the species generated by Type I and Type II mechanisms have opposite effects on proteasome activity, being Type I a positive activator while Type II lead to impairment of proteasome function.     

Inhibition of protein tyrosine phosphatases by amino acid, peptide, and protein hydroperoxides: potential modulation of cell signaling by protein oxidation products

Reaction of radicals in the presence of O2, or singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. They can be detected in cells and are poorly removed by enzymatic defenses. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, with this resulting in inactivation of some thiol-dependent enzymes. In light of these data, we hypothesized that inactivation of protein tyrosine phosphatases (PTPs), by hydroperoxides present on oxidized proteins, may contribute to cellular and tissue dysfunction by modulation of phosphorylation-dependent cell signaling. We show here that PTPs in cell lysates, and purified PTP-1B, are inactivated by amino acid, peptide, and protein hydroperoxides in a concentration- and structure-dependent manner. Protein hydroperoxides are particularly effective, with inhibition occurring with greater efficacy than with H2O2. Inactivation involves reaction of the hydroperoxide with the conserved active-site Cys residue of the PTPs, as evidenced by hydroperoxide consumption measurements and a diminution of this effect on blocking the Cys residue. This inhibition of PTPs, by oxidized proteins containing hydroperoxide groups, may contribute to cellular dysfunction and altered redox signaling in systems subject to oxidative stress.     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

A synthetic defect in protein degradation caused by loss of Ufd4 and Rad23

The UFD (ubiquitin fusion degradation) pathway is responsible for multiubiquitination of the fusion proteins that bear a "non-removable" N-terminal ubiquitin moiety. Previous reports have shown that the UFD pathway is conserved from yeast to human. The essential elements of the UFD pathway have also been identified in Saccharomyces cerevisiae. These studies, however, are limited to use of engineered UFD substrates. The biological significance of the UFD pathway remains unknown. Here we demonstrate that Ufd4, the E3 component of the UFD pathway, is involved in controlling the degradation of Rad4, a nucleotide excision repair protein. Moreover, simultaneous loss of Ufd4 and Rad23 exhibits a synthetic inhibitory effect on Rad4 degradation, presenting the first example that a UBA/UBL-domain protein functionally overlaps with a ubiquitin ligase in determining the turnover rate of a protein substrate. The current work also provides a direction for further investigation of the physiological functions of the UFD pathway.     

Variation of frequency spectrum of the erythrocyte flickering caused by aging,osmolarity, temperature and pathological changes

Frequency spectra of the surface undulations (flickering) of erythrocyte plasma membranes are measured by direct spectral analysis of the intensity fluctuations of the light passing the cells in a phase contrast microscope. Spectra are taken as a function (1) of the temperature (2) of the viscosity and osmolarity of the outer medium (3) of the aging of cells and (4) of pathological transformations. The spectra are approximately superpositions of two Lorentzian lines. At large frequencies,f, the spectra follow f^?2. This behaviour can be interpreted in terms of cell thickness fluctuations caused by thermally excited membrane undulations provided the range of wavelengths is small. The undulations are determined by the membrane curvature elasticity while the lateral tension is negligibly small for cells of discoid shape. The technique presented allows accurate measurements of relative curvature (bending) elastic constants. The spectra of freshly drawn cells are remarkably reproducible. Aging of the cells in the medium leads to an increase in the curvature elastic constant. A decrease in osmolarity causes a reduction in the intensity and line width of the spectra and the flickering vanishes if the cell approaches a spherical shape. The effect of temperature between 10 and 40°C is astonishingly small with the exception of a sudden increase in the amplitude with increasing temperature at 35°C. The flicker spectra of a large fraction of the cells from patients suffering from cronical alcoholism exhibit a reduced line width or an increase in the curvature elastic constant.     

Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies: preparation for a functional analysis

Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0M urea in 0.5M increments. Purified RHG1 protein obtained from the 1.5 and 7.0M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0M urea sample, whereas refolding from the 1.5M urea sample yielded 0.2mg/ml protein. The 7.0M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.     

Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition

Mutations in DJ-1 gene have been linked to autosomal recessive early onset parkinsonism (AR-EOP). Although the mechanism of neuronal cell death due to DJ-1 mutation has not been fully elucidated, loss of DJ-1 function was considered to cause the phenotype. Here, we demonstrated that the down regulation of endogenous DJ-1 of the neuronal cell line by siRNA enhanced the cell death which was induced by oxidative stress, ER stress, and proteasome inhibition, but not by pro-apoptotic stimulus. The cell death with hydrogen peroxide was dramatically rescued by over-expression of wild-type DJ-1, but not by that of L166P mutant DJ-1. Furthermore, DJ-1 rescued the cell death caused by over-expression of Pael receptor, which was a substrate of Parkin, another gene product for autosomal recessive juvenile parkinsonism. These results suggest that loss of protective activity of DJ-1 from neuro-toxicity induced by these stresses contributes to neuronal cell death in AR-EOP with mutant DJ-1.