Protein degradation

As cells age, detritus inevitably accumulates; one theory of aging holds that rising levels of unwanted molecules will eventually become cytotoxic — the so-called “garbage catastrophe” model — and this, in turn, could cause age-related decline in cell and tissue function.

Cells have a variety of means to eliminate misfolded, damaged and covalently altered proteins, among them the ubiquitin-proteasome pathway and the various flavors of autophagy. These mechanisms of cellular trash collection have come under increasing scrutiny by biogerontologists — and the results, while generally consistent with the idea that protein recycling is important in the aging process, can often be surprising: in some organ systems (like the brain) excessive autophagy can be deleterious. And in prematurely aging mice, the most recent observations are somewhat counter-intuitive:

Activation of autophagy in progeria: Autophagy and aging: New lessons from progeroid mice, Mariño y López-Otín:

We have recently reported the unexpected finding that distinct progeroid murine models exhibit an extensive basal activation of autophagy instead of the characteristic decline in this process occurring during normal aging. … [T]he observed autophagic increase is associated with a series of metabolic alterations resembling those occurring under calorie restriction or in other situations reported to prolong lifespan.

Regulation by circadian clocks: Diurnal rhythms of autophagy: Implications for cell biology and human disease, Sachdeva and Thompson:

As a consequence of the induction of autophagy during short periods of fasting, animals experience diurnal rhythms of autophagy in concert with their circadian cycle. … Whether the circadian clock directly regulates autophagy in mammalian cells, or whether autophagy may play a role in the cycling of mammalian cell clocks is not yet clear. Nevertheless, the relationship between circadian cycles and autophagy is an intriguing area for future study and has implications for multiple human diseases, including aging, neurodegeneration, and cancer.

Life extension therapeutics?: Proteasome activation as a novel antiaging strategy, Chondrogianni and Gonos:

As proteasome has an impaired function during aging, emphasis has been given recently in identifying ways of its activation. A number of studies have shown that the proteasome can be activated by genetic manipulations as well as by factors that affect its conformation and stability. Importantly the developed proteasome activated cell lines exhibit an extended lifespan. … Finally as few natural compounds have been identified having proteasome activation properties, we discuss the advantages of this novel antiaging strategy.

Protein misfolding in neuropathological states: Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging, Rick Morimoto:

Adaptation and survival requires the ability to sense damaged proteins and to coordinate the activities of protective stress response pathways and chaperone networks. Yet, despite the abundance and apparent capacity of chaperones and other components of homeostasis to restore folding equilibrium, the cell appears poorly adapted for chronic proteotoxic stress when conformationally challenged aggregation-prone proteins are expressed in cancer, metabolic disease, and neurodegenerative disease. The decline in biosynthetic and repair activities that compromises the integrity of the proteome is influenced strongly by genes that control aging, thus linking stress and protein homeostasis with the health and life span of the organism.

Looks like tomorrow will be a big one on mitochondria and oxidative damage.


Once again the booming literature on calorie restriction (CR) has bested me, and I’ve fallen hopelessly behind. Therefore, without comment, I’ll just run through the last month’s abstracts, with a smattering of brief commentary here and there. Each paper deserves its own entry, but we’re just going to have to make do with this. Quoted passages are all abstract excerpts.

The Nrf2 pathway: Mechanisms Underlying Caloric Restriction and Lifespan Regulation: Implications for Vascular Aging, Ungvari et al.:

We propose that caloric restriction increases bioavailability of NO, decreases vascular reactive oxygen species generation, activates the Nrf2/antioxidant response element pathway, inducing reactive oxygen species detoxification systems, exerts antiinflammatory effects, and, thereby, suppresses initiation/progression of vascular disease that accompany aging.

More on Nrf2 and aging here and here.

Protein vs. sugar in insulin signaling: Opposing Effects of Dietary Protein and Sugar Regulate a Transcriptional Target of Drosophila Insulin-like Peptide Signaling, Buch et al.

Through microarray analysis of flies in which the insulin-producing cells (IPCs) were ablated, we identified a target gene, target of brain insulin (tobi), that encodes an evolutionarily conserved -glucosidase. Flies with lowered tobi levels are viable, whereas tobi overexpression causes severe growth defects and a decrease in body glycogen. Interestingly, tobi expression is increased by dietary protein and decreased by dietary sugar.

Inactivity and inflammation: Calorie restriction modulates inactivity-induced changes in the inflammatory markers CRP and PTX3, Busutti et al.:

Calorie restriction prevents the inflammatory response induced by 14 days of bed rest. We suggest an inverse regulation of CRP and PTX3 in response to changes in energy balance.

*** This was a human study.

“Nutritional emphysema”: Effect of Severe Calorie Restriction on the Lung in Two Strains of Mice, Bishai and Mitzner:

Although the baseline mechanics and alveolar size were quantitatively different in the two strains, both strains showed similar qualitative changes during the starvation and refeeding periods. Thus, in two strains of mice with genetically determined differences in alveolar size neither the mechanics nor the histology show any evidence of emphysema-like changes with this severe caloric insult.

SIRT1 stabilization: Regulation of SIRT1 protein levels by nutrient availability, Kanfi et al.:

We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions.

Protein modification in the heart: Aging and dietary restriction effects on ubiquitination, sumoylation, and the proteasome in the heart, Li et al.:

Cumulatively, our data indicate that DR has many beneficial effects towards the UPP [ubiquitin-proteasome pathway] in the heart, and suggests that a preservation of the UPP may be a potential mechanism by which DR mediates beneficial effects on the cardiovascular system.

Males vs. females, round 1: The brain: Conserved and Differential Effects of Dietary Energy Intake on the Hippocampal Transcriptomes of Females and Males, Martin et al.:

Genes involved in energy metabolism, oxidative stress responses and cell death were affected by the HFG diet in both males and females. The gender-specific molecular genetic responses of hippocampal cells to variations in dietary energy intake identified in this study may mediate differential behavioral responses of males and females to differences in energy availability.

Males vs, females, round 2: The gonad: Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary, Sharov et al.:

CR-mediated reversal of age-associated gene expression changes, reported in somatic organs previously, was limited to a small number of genes in gonads. Instead, in both ovary and testis, CR caused small and mostly gonad-specific effects: suppression of ovulation in ovary and activation of testis-specific genes in testis.

Whew. OK, have a great weekend, everyone.

The proteasome is an essential agent in cellular protein turnover, recognizing and targeting polyubiquitinated proteins for degradation — a process that serves both a regenerative function (by turning proteins back into amino acids, the raw materials for proteins yet to be synthesized) and a regulatory one (a protein that has been degraded can no longer act within the cell).

Papa and Rockwell report that reversible proteasome inhibition has long-term effects on the mitochondria, causing diminished energy production and increased generation of reactive oxygen species (ROS) in neurons. This increase in oxidative stress, in turn, results in increased cell death via apoptosis:

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored.

Here’s what caught my eye about this paper: Mitochondrial ROS production is widely considered to play a causative role in cellular aging. Mitochondria, in turn, accumulate damage over the lifespan, which causes an acceleration in ROS production — a nasty positive-feedback loop. Based on these findings, however, I wonder whether the decline in mitochondrial function could be driven not only from within (by oxidative damage to mitos causing further oxidation), but also from without: We know that proteasome efficiency also declines during aging; it would seem likely that this functional decline could further erode mitochondrial function.

One corollary of this hypothesis is that even if we were able to completely eliminate oxidative damage, we’d still suffer diminution in mitochondrial capacity as a result of proteasomal decline –unless, of course, this decline and its effect on mitochondria operate via a non-oxidative mechanism, in which case eliminating oxidative damage would kill two birds with one stone.

The cellular housekeeping program known as autophagy — which allows the recycling of old (and potentially damaged) proteins, lipids, and even whole organelles — is essential to maintaining cellular health over long periods of time. Recent findings implicate autophagy in the regulation of lifespan: Without autophagy, damaged macromolecules will slowly accumulate over time, potentially resulting in a breakdown of cellular homeostasis termed a garbage catastrophe — and unfortunately, it appears that the efficiency of autophagy decreases with age.

Two recent articles underscore the importance of autophagy in the longevity of two favorite model systems: the fly Drosophila and the worm C. elegans. Juhász and Neufeld report that fly mutants in the autophagy gene Atg7 are viable and develop normally, but are hypersensitive to oxidation and starvation stress (two hallmarks of premature aging mutants) and undergo premature neuronal cell death. These results are consistent with earlier findings linking defects in lysosomal trafficking (the intracellular sorting mechanism that guides the targets of autophagy to their ultimate fate) to shortened longevity, stress resistance and phenotypes of premature aging.

Meanwhile, in the worm (where, autophagy is a two-edged sword, at least in the “brain”) Tóth et al. describe how multiple longevity assurance pathways (IGF-1 and TOR) converge on autophagy. They argue that the regulation of autophagy represents a key mechanism by which these pathways regulate longevity — a model that makes a large number of strong predictions about genetic interactions between these pathways (predictions which, one hopes, the authors are currently hard at work testing).

After yesterday’s feast of autophagy I realized that I have another full meal’s worth of stories regarding chaperones and heat shock proteins (HSPs). Regulation of chaperone genes — which catalyze folding and assembly of proteins, and also help dispose of malfolded proteins — already holds a special place in biogerontologists’ hearts: the life-extending compound resveratrol induces the heat shock response (possibly in an example of hormesis), and stochastic changes in chaperone levels are associated with parallel changes in organismal lifespan.

Without further ado then, here are three recent articles on chaperones and heat shock:

  • Just as autophagy declines with age, so too does the activity of the proteasome and the expression of the major cytosolic chaperone Hsc70. Bonelli et al. describe how calorie restriction (CR) of rats maintains proteasome activity and Hsc70 expression in the liver, well into old age. In contrast to CR’s effects on autophagy, the boost in proteasome activity does not appear to occur simply as a result of increased expression of proteasome components; the authors argue that something is acting to qualitatively increase the activity of proteasomes while keeping their levels unchanged. (Maybe targeting or delivery is more efficient?)
  • In humans, several HSPs are upregulated with age, perhaps as a response to increased steady-state levels of oxidatively damaged proteins, which would be expected to fold poorly. Njemini et al. describe an interesting association of increased HSP expression with the increased inflammatory cytokine expression that is a hallmark of frailty in old age.
  • In budding yeast, daughter cells are protected from inheriting their mother cells’ old age because the mothers keep damaged macromolecules on their side of an asymmetric cell division, a process that requires SIR2, the founding member of the sirtuin family. Erjavec et al. demonstrate that the sequestration of oxidatively altered proteins also requires Hsp104. Overproduction of this chaperone can rescue the accelerated-aging phenotype of sir2 mutants.

Autophagy is increasingly thought to play an important role in aging, in part because autophagic degradation allows cells to prevent the accumulation of damaged macromolecules such as oxidized proteins and advanced glycation endproducts (AGEs).

I’ve been slowly squirreling away abstracts about the subject, and they’ve been building up — so on this crisp nearly-winter day I figured that it might be time to dig up some of those nuts:

  • In heart muscle, calorie restriction (CR) boosts autophagy, both by increasing the number of autophagosomes and the expression of autophagy-related proteins. Wohlgemuth et al. suggest that this might provide a mechanism for the beneficial effects of CR on cardiac tissue.
  • The overall rate of autophagy decreases with age. One specific flavor of degradation that undergoes such a decline is chaperone-mediated autophagy, which directs cytosolic proteins to the lysosome via an interaction with an intracellular receptor. Kiffin et al. have tracked down the cause of this decline, and lay the blame on changes in the processing and stability of the lysosomal receptor LAMP-2.
  • The benefits of autophagy (and the hazards resulting from its age-related decline) are hinted at in the results of Bergamini et al., who show that an anti-lipolytic drug that stimulates autophagy can reverse at least one biomarker of aging, in this case high levels of altered mitochondrial DNA. The authors also address the relationship between CR and the cellular adaptation to fasting, and argue that autophagy is essential to the anti-aging action of CR.

Following on the heels of comprehensive reviews of mitochondria, senescence and cancer as they relate to aging, Vernace et al a nice overview of the role of protein degradation in the process. Motivated by recent findings in neurodegenerative disease, they focus on the possibility that dwindling efficiency of the ubiquitin/proteasome pathway may limit cellular lifespan:

Aging and regulated protein degradation: who has the UPPer hand?
In all cells, protein degradation is a constant, ongoing process that is critical for cell survival and repair. The ubiquitin/proteasome pathway (UPP) is the major proteolytic pathway that degrades intracellular proteins in a regulated manner. It plays critical roles in many cellular processes and diseases. Disruption of the UPP is particularly relevant to pathophysiological conditions that provoke the accumulation of aberrant proteins, such as in aging as well as in a variety of neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases. For unknown reasons, most of these neurodegenerative disorders that include familial and sporadic cases exhibit a late onset. It is possible that these neurodegenerative conditions exhibit a late onset because proteasome activity decreases with aging. Aging-dependent impairment in proteolysis mediated by the proteasome may have profound ramifications for cell viability. It can lead to the accumulation of modified, potentially toxic proteins in cells and can cause cell injury or premature cell death by apoptosis or necrosis. While it is accepted that aging affects UPP function, the question is why does aging cause a decline in regulated protein degradation by the UPP? Herein, we review some of the properties of the UPP and mechanisms mediating its age-dependent impairment. We also discuss the relevance of these findings leading to a model that proposes that UPP dysfunction may be one of the milestones of aging.

In addition to simply allowing misfolded proteins to accumulate, of course, alterations in the pathway will also result in changes to the half-lives of proteins that are regulated by ubiquitin-mediated degradation; this could also have important ramifications in the biology of aging (see Ubiquitin, the proteasome, and aging).

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