Protein degradation

(previous session)

Craig Skinner (Lin Lab, UC Davis): Identification of potential calorie restriction mimics in yeast using a nitric oxide-based screen. Yeast are an important model system in biogerontology, useful not only for genetic studies of longevity control but also for discovery of bioactive compounds. Calorie restriction (CR) in yeast causes increased levels of nitric oxide (NO) — somewhat surprising in that yeast cells lack a homolog of nitric oxide synthase — and elevated NO is sufficient to extend yeast lifespan. These observations led Skinner to screen a yeast deletion library for elevated NO levels, yielding several genes that extend lifespan.

Mark Lucanic (Lithgow Lab, Buck): Endocannabinoid signaling mediates the effect of diet on lifespan in C. elegans. Mutants in the dauer pathway in C. elegans often influence longevity; the daf-2 mutation, which causes constitutive dauer formation at elevated temperatures, extends lifespan by several fold. Lucanic discovered that endocannabinoids are involved in the regulation of the dauer pathway — and therefore, of longevity — either independently of or far downstream of daf-2 and daf-16. Endocannabinoids are upregulated under well-fed conditions, and shorten lifespan.

Delia David (Kenyon Lab, UCSF): Widespread protein aggregation is an inherent part of aging in C. elegans. Protein aggregates are a hallmark of many age-related neurodegenerative diseases, leading to the hypotheses that the cellular mileu changes with age in a manner that causes native, aggregation-prone proteins to form aggregates. David used mass spectrometry to identify a subset of normal worm proteins aggregate as a function of age. As with the proteins associated with neurodegeneration, specific proteins aggregate in specific cell types. Mutations that extend lifespan (such as daf-2) decrease aggregation, and tend to downregulate the expression of genes encoding aggregation-prone proteins. Curiously, regulators of protein homeostasis tend to aggregate themselves, leading to a destructive positive feedback loop in which the very factors that protect the cell from proteotoxicity disappear into aggregates, leading to further aggregation.

Cherry Tang (Zhong Lab, Berkeley): The Clearance of Ubiquitinated Protein Aggregates Via Autophagy. Autophagic protein degradation has been implicated in control of lifespan: autophagy slows cell and tissue aging. Tang has identified a protein that participates in degradation of ubiquitinated proteins and co-localizes with autophagosomes; when the protein is knocked down, protein aggregates become more toxic.

(next session)


Cells tend to produce unwanted protein aggregates and other molecular refuse slightly faster than they can get rid of it, resulting in a time-dependent accumulation of potentially toxic cellular garbage. This, in turn, can cause an age-dependent loss of cellular viability, which is (in certain contexts) a fair operational definition of aging.

How can cells deal with their garbage? Protein aggregates are both sticky and insoluble, making it hard for cellular machinery to deal with them at an enzymatic level. If the gunk can’t be eliminated, however, it might still be possible to move it around in a useful way. Specifically, at mitosis, the cell could make sure that all the potentially toxic aggregates stay in one of the progeny. To illustrate the argument I’ll turn to the words of the estimable Alex Palazzo:

One approach is to distribute everything equally amongst your two offspring. …

A second approach is to give all the crap to one of the two new cells and keep the other one pristine. Lets call these two cells the crap cell and the pristine cell. What’s the result of this second strategy? Using our crap metric from above, the first cell accumulates 10 units of garbage over its lifetime and then gives it all to one offspring, the crap cell, and none to the other offspring, the pristine cell. Those cells then grow and by the time they divide each second generation cells have made 10 units of additional crap each. The crap cell has 20 units the pristine cell 10. The two cells divide and dump all their garbage on one of their offsprings. One cell starts with 20 units of crap, one cell with 10 units and two cells are again crap free. The end result of this strategy? Part of your descendents will become more and more decrepit as they fill up with crap, while others remain pristine.

The crap cell (I love this nomenclature) will become inviable sooner under this strategy, but the alternative would be a symmetric division strategy in which all descendants accumulate garbage, ultimately causing the extinction of the entire lineage. The idea here is that assuming certain values for adjustable parameters re: the rate of garbage accumulation and the effect of garbage level on reproductive fitness, this can be an advantageous strategy to ensure reproductive success. Both single-celled yeast and mammalian stem cells employ this asymmetric strategy in order to preserve the viability of an indefinitely dividing lineage.

In yeast, the crap cell is called the “mother”; the pristine cell is called the “daughter” — mom accumulates garbage of various kinds, both protein aggregates and rDNA circles. When the mother is ready to divide, a bud forms at a specific site on her cell wall, defined by a set of macromolecular complexes that determine cellular polarity. Liu et al. have demonstrated that the daughter cell is using some of the same polarity-determining machinery (the “polarisome”) to actively transport protein aggregates back into the mother:

The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates

The paradigm sirtuin, Sir2p, of budding yeast is required for establishing cellular age asymmetry, which includes the retention of damaged and aggregated proteins in mother cells. By establishing the global genetic interaction network of SIR2 we identified the polarisome, the formin Bni1p, and myosin motor protein Myo2p as essential components of the machinery segregating protein aggregates during mitotic cytokinesis. Moreover, we found that daughter cells can clear themselves of damage by a polarisome- and tropomyosin-dependent polarized flow of aggregates into the mother cell compartment. The role of Sir2p in cytoskeletal functions and polarity is linked to the CCT chaperonin in sir2Δ cells being compromised in folding actin. We discuss the findings in view of recent models hypothesizing that polarity may have evolved to avoid clonal senescence by establishing an aging (soma-like) and rejuvenated (germ-like) lineage.

Note the role for Sir2p, the founding member of the sirtuin family of longevity assurance genes: Sir2p is required, via another protein’s activity, for the normal folding of actin, the cytoskeletal protein from which the daughter-mother transport cable is built. It’s an indirect interaction, and more complex than I’m making it out to be here. Nonetheless, it is satisfying for those of us looking for unifying theories in aging that one of the most widely studied proteins in lifespan regulation is involved in the deep connection between polarity and aging.

I’ll close with a few questions:

  • Why can’t the mother cell export the aggregates? One of our initial premises was that aggregates are biochemically hard to handle, which is why they accumulate rather than being degraded. But now we know that cells can bundle aggregates onto actin cables and move them around — why not sort the aggregates into vesicles or membrane blebs and dispose of them? Granted, in order to export an aggregate out of the cell, it would have to cross a membrane, but this would be no more difficult topologically than mitophagy. The obvious (and trivial) answer to this question is “because it didn’t evolve that way,” but I’m curious to know whether there’s some compelling reason why it couldn’t have evolved that way.
  • How do symmetrically dividing cells overcome this problem? In order to exploit asymmetric division, one must first establish polarity. The argument above about the rate of garbage accumulation would seem to apply equally well to non-polarized cells like bacteria – why, then, do clonal lineages of symmetrically dividing cells not invariably go extinct? Maybe the cells that we think are symmetric are secretly asymmetric, with a crap/pristine segregation that has yet to be uncovered. Or maybe the symmetric cells know something about garbage disposal that we don’t. In either case, there’s something important to learn that might help us keep mammalian cells youthful.

ResearchBlogging.orgLiu, B., Larsson, L., Caballero, A., Hao, X., Öling, D., Grantham, J., & Nyström, T. (2010). The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates Cell, 140 (2), 257-267 DOI: 10.1016/j.cell.2009.12.031

The TOR (“target of rapamycin”) protein is a master regulator of cell growth, governing connect nutrient sensing, protein synthesis, and proliferation. It has become increasingly clear that the TOR pathway plays an essential role in longevity determination — specifically, higher TOR activity is associated with more rapid aging and shorter lifespan.

In mammals, TOR interferes with stem cell functions, and TOR activity is downregulated by exercise. It has been proposed that TOR inhibitors might even be used as anti-aging drugs (and in fact we’re going to investigate some recent relevant tests of that idea, sometime next week). The relationship between TOR and lifespan holds true across great evolutionary distances: loss of TOR function (in conjunction with other mutations) can dramatically increase the chronological lifespan of yeast.

How does TOR control the rate of aging? In order to answer this question, we must look downstream, to proteins that are controlled by TOR. A recent study from the Kapahi lab (our neighbors at the Buck Institute for Age Research) investigated the role of one such TOR target: HIF-1 (“hypoxia inducible factor”; it is also involved in metabolism). The authors find that loss-of-function mutations in HIF-1 result in longer-lived C. elegans. Chen et al.:

HIF-1 Modulates Dietary Restriction-Mediated Lifespan Extension via IRE-1 in Caenorhabditis elegans

Dietary restriction (DR) extends lifespan in various species and also slows the onset of age-related diseases. Previous studies from flies and yeast have demonstrated that the target of rapamycin (TOR) pathway is essential for longevity phenotypes resulting from DR. TOR is a conserved protein kinase that regulates growth and metabolism in response to nutrients and growth factors. While some of the downstream targets of TOR have been implicated in regulating lifespan, it is still unclear whether additional targets of this pathway also modulate lifespan. It has been shown that the hypoxia inducible factor-1 (HIF-1) is one of the targets of the TOR pathway in mammalian cells. HIF-1 is a transcription factor complex that plays key roles in oxygen homeostasis, tumor formation, glucose metabolism, cell survival, and inflammatory response. Here, we describe a novel role for HIF-1 in modulating lifespan extension by DR in Caenorhabditis elegans. We find that HIF-1 deficiency results in extended lifespan, which overlaps with that by inhibition of the RSKS-1/S6 kinase, a key component of the TOR pathway. Using a modified DR method based on variation of bacterial food concentrations on solid agar plates, we find that HIF-1 modulates longevity in a nutrient-dependent manner. The hif-1 loss-of-function mutant extends lifespan under rich nutrient conditions but fails to show lifespan extension under DR. Conversely, a mutation in egl-9, which increases HIF-1 activity, diminishes the lifespan extension under DR. This deficiency is rescued by tissue-specific expression of egl-9 in specific neurons and muscles. Increased lifespan by hif-1 or DR is dependent on the endoplasmic reticulum (ER) stress regulator inositol-requiring protein-1 (IRE-1) and is associated with lower levels of ER stress. Therefore, our results demonstrate a tissue-specific role for HIF-1 in the lifespan extension by DR involving the IRE-1 ER stress pathway.

The mutants’ life extension was observed when the worms could eat ad libitum but not when they were dietarily restricted (DR), implying that the mechanism of the HIF-1 mutation is similar to that of DR. Conversely, activation of HIF-1 expression (by mutating EGL-9, which ubiquitinates HIF-1) decreases the lifespan extension due to DR. Taken together, the findings imply that downregulation of HIF-1 expression is both necessary and sufficient for DR-mediated longevity enhancement.

One more step down the rabbit hole, then: What are HIF-1 and DR doing? The authors find that lifespan extension requires the IRE1-gene, a principle mediator of the unfolded protein response (UPR). The UPR is activated when the endoplasmic reticulum (ER) is stressed — when protein folding is inefficient, or the secretory machinery is overloaded; the pathway returns the cell to homeostasis by inducing expression of genes that fold, sort, and process proteins in the ER (or degrade the proteins that can’t be saved). Perhaps lifespan extension requires increased ER capacity, or more efficient degradation of misfolded proteins?

On a closing note: Attentive readers will have recalled that not very long ago, we reported on a paper that appears to have reached the opposite conclusion — specifically, that high expression of HIF-1 (induced the same way as here, by mutation in the ubiquitin E3 ligase EGL-9) results in extended lifespan and decreased proteotoxicity. I don’t want to get in the middle of this controversy, except to point out that the systems were different in a number of ways, and that it is a formal possibility that a gene’s activity could be “tuned” such that either an increase or a decrease in expression could increase lifespan (implying that the wildtype expression levels are at a “sweet spot” of lower lifespan but presumably higher fitness, due to some sort of tradeoff between longevity and reproductive success). I am sure that the authors of both studies are working to reconcile the apparent contradiction. We’ll look forward to learning more as the story develops.

ResearchBlogging.orgChen, D., Thomas, E., & Kapahi, P. (2009). HIF-1 Modulates Dietary Restriction-Mediated Lifespan Extension via IRE-1 in Caenorhabditis elegans PLoS Genetics, 5 (5) DOI: 10.1371/journal.pgen.1000486

How do cells get rid of their garbage? One solution is simply to outgrow it: if the rate of synthesis of new components exceeds the rate of accumulation of old ones, then unwanted trash will be diluted out, even without any active clearance. This is only really possible in exponentially growing populations, however: slowly dividing or postmitotic cells must activate degradative pathways (e.g., autophagy and ubiquitin-proteasome) in order to prevent accumulation of potentially toxic damaged macromolecules and dysfunctional organelles.

In order to degrade a protein, protein complex or organelle, however, one must first be able to get at it — and there are specific cellular components that make this very difficult. As an example, let’s consider the nuclear pore complex (NPC): it’s huge (120 megaDaltons), complex (>30 protein components) and topologically challenging (the pore crosses the nuclear envelope and creates a hole in the process). Many NPC components aren’t in dynamic equilibrium with cytosolic pools, so if we want to turn over any of these proteins we would have to somehow take out the entire NPC, repair the ensuing damage to the membrane, and then either re-insert the NPC (which I don’t believe actually happens) or synthesize a new NPC to restore the lost import/export capacity.

Unfortunately, new nuclear pores are probably only created during mitosis, when the nuclear envelope and topologically connected endoplasmic reticulum (ER) split up into vesicles that subsequently re-fuse after cell division is complete. So how do postmitotic cells turn over and degrade NPCs?

The answer, according to D’Angelo et al., is that they probably don’t. Instead, NPCs get old, and accumulate damage, and eventually stop doing their job:

Age-Dependent Deterioration of Nuclear Pore Complexes Causes a Loss of Nuclear Integrity in Postmitotic Cells

In dividing cells, nuclear pore complexes (NPCs) disassemble during mitosis and reassemble into the newly forming nuclei. However, the fate of nuclear pores in postmitotic cells is unknown. Here, we show that NPCs, unlike other nuclear structures, do not turn over in differentiated cells. While a subset of NPC components, like Nup153 and Nup50, are continuously exchanged, scaffold nucleoporins, like the Nup107/160 complex, are extremely long-lived and remain incorporated in the nuclear membrane during the entire cellular life span. Besides the lack of nucleoporin expression and NPC turnover, we discovered an age-related deterioration of NPCs, leading to an increase in nuclear permeability and the leaking of cytoplasmic proteins into the nucleus. Our finding that nuclear leakiness is dramatically accelerated during aging and that a subset of nucleoporins is oxidatively damaged in old cells suggests that the accumulation of damage at the NPC might be a crucial aging event.

The damaged NPCs are no longer effective at actively segregating cytosolic and nuclear proteins; the resulting “leakiness” exemplifies the general principle of age-related loss of fidelity. This leakiness could be causally connected to aging in several different ways: components targeted to the wrong compartment could have deleterious consequences in their new homes; damaged NPCs could distort the nuclear envelope in a manner analogous to the effect of lamin mutations; or a combination of these and other effects could contribute to the transcriptional dysregulation that has been observed in multiple cell types and experimental systems.

The fidelity of protein synthesis is necessary for a properly functioning organism. In an aged animal, the overall rates of protein synthesis and degradation/recycling decline with age. Not only does this decrease the number of structural and enzymatic proteins available, but it increases the half life of proteins, perhaps allowing more time for these proteins to become oxidatively damaged.

Protein synthesis is also linked to nutrient availability through the TOR signaling pathway, implicating protein synthesis in the mechanism of life extension by calorie restriction (CR). CR counteracts the decline in protein turnover seen with age. Inhibiting protein synthesis also increases lifespan in C. elegans.

Wang et al examined the link between the rate of protein synthesis and mitochondrial degeneration. Working in yeast, they introduced a mutation into adenine nucleotide translocase (aac2A128P), a protein located on the mitochondrial membrane. This mutation mimics the human disease progressive external ophthalmoplegia (PEO). The authors speculate that aac2A128P might upset the availability of nucleotides, which in turn could cause deletions of mtDNA, a hallmark of PEO. In addition, they show that the introduction of aac2A128P causes a decrease in membrane potential, which has already been shown to play a key role in aged mitochondria. aac2A128P mutants also have a decreased replicative lifespan.

The authors next tested whether any known lifespan-extending mutations can suppress the mitochondrial dysfunction induced by the aac2A128P mutation. Three of these mutations were particularly robust in reversing the aac2A128P mutation. sch9Δ, rpl6BΔ and rei1Δ on a aac2A128P background were able to form viable colonies at almost wild type levels. SCH9 is involved in nutrient sensing; RPL6B is a protein that makes up the ribosome; and REI1 is involved in ribosome processing. Overexpression of the well-known yeast sirtuin SIR2 did not have any effect on replicative lifespan in the aac2A128P mutant.

sch9Δ, rpl6BΔ and rei1Δ mutants, even with the aac2A128P mutation, outlived their wild type counterparts. The lethal aac2A128P/phb1Δ double mutant was still viable with these three life-extending mutations. phb1Δ mutants have reduced mitochondrial membrane potentials and are susceptible to ethidium bromide. sch9Δ, rpl6BΔ and rei1Δ mutants on a phb1Δ background were resistant to ethidium bromide. CR had similar effects as these three lifespan-extending mutants, but other long-lived mutants, like tor1Δ, were only able to suppress the aac2A128P – and not the aac2A128P/phb1Δ double mutant – defect in mitochondrial membrane potential.

Might mitochondrial membrane potential be linked to protein synthesis? The researchers found that all of the lifespan-extending mutations examined had reduced total protein synthesis. sch9Δ, rpl6BΔ and rei1Δ mutants had some of the largest decreases. According to the authors, lower rates of protein translation will reduce stress from unassembled protein complexes. “Reduction of cytosolic protein synthesis may lower the overall loading of proteins onto the mitochondrial inner membrane and promote mitochondrial membrane potential maintenance.”

To examine the link between mitochondrial membrane potential and protein synthesis, the authors looked at the yme1Δ mutant. YME1 encodes a protease important for protein turnover, so, according to the authors, the altered mitochondrial membrane potential of the aac2A128P mutant will worsen with decreased protein turnover. sch9Δ, rpl6BΔ and rei1Δ mutants were able to suppress the lethal aac2A128P/yme1Δ double mutant.

This paper links two aging-associated deficiencies: mitochondrial defects and decreases in protein synthesis. These connections are becoming more and more common in the aging field. But why did some life-extending mutations cause reversions in the aac2A128P mutant while others didn’t? How many paths are there to extended longevity?

The data above presents another conundrum. Protein synthesis declines with age, yet decreasing protein synthesis extends lifespan. What’s going on here? My guess is that decreased protein synthesis when you’re young in addition to inhibiting the decline in protein translation as you get older will delay the aging process.

Continuing with the theme of unifying theories in aging: Over at The Daily Transcript, Alex Palazzo has written up a thoughtful and detailed analysis of a recent review (the original paper is entitled Polarity and Differential Inheritance: Universal Attributes of Life?). In his post, he also introduces some fantastic new nomenclature (emphasis in the original).

Let’s pretend you are a unicellular organism – what would be the best strategy to ensure the long-term multi-generational survival of your lineage?

One approach is to distribute everything equally amongst your two offspring. …

A second approach is to give all the crap to one of the two new cells and keep the other one pristine. Lets call these two cells the crap cell and the pristine cell. What’s the result of this second strategy? Using our crap metric from above, the first cell accumulates 10 units of garbage over its lifetime and then gives it all to one offspring, the crap cell, and none to the other offspring, the pristine cell. Those cells then grow and by the time they divide each second generation cells have made 10 units of additional crap each. The crap cell has 20 units the pristine cell 10. The two cells divide and dump all their garbage on one of their offsprings. One cell starts with 20 units of crap, one cell with 10 units and two cells are again crap free. The end result of this strategy? Part of your descendents will become more and more decrepit as they fill up with crap, while others remain pristine.

Somewhat reminiscent of the garbage catastrophe, but with important differences — and this time with evolutionary legs.

Alex has teased us with the suggestion that this will be the first of multiple parts to his coverage of this issue. If so, that’s exciting news; he has done some excellent multi-part stories in the past (e.g. these posts on cancer and metabolism). I’m looking forward to the next chapter.

As cells age, they accumulate junk: cross-linked proteins, oxidized lipids, dysfunctional membrane-bound organelles, and other detritus. Such undesirables can interfere, directly or indirectly, with the proper functioning of the cell; therefore, it’s important for cells to have ways to take out the trash. One important class of garbage-disposal pathways is collectively termed autophagy (etymologically, “self-eating”); together, these mechanisms help to clear the cell of damaged macromolecules.

The rate of autophagy slows with age, making turnover of damaged components less efficient. This could conceivably set up a vicious cycle: slower autophagy –> more rapid accumulation of toxic protein aggregates –> further interference with cellular machinery, including autophagy itself –> even slower autophagy. This model has been termed the “garbage catastrophe” hypothesis, and is a prime contender for explaining age-related decline in cellular function.

The model makes a strong prediction: if one were able to prevent the age-associated decline in autophagy, one could delay or prevent this functional decline. That prediction was successfully put to the test by Zhang and Cuervo, who created a transgenic mouse whose autophagic activity can be experimentally modulated:

Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function

Chaperone-mediated autophagy (CMA), a selective mechanism for degradation of cytosolic proteins in lysosomes, contributes to the removal of altered proteins as part of the cellular quality-control systems. We have previously found that CMA activity declines in aged organisms and have proposed that this failure in cellular clearance could contribute to the accumulation of altered proteins, the abnormal cellular homeostasis and, eventually, the functional loss characteristic of aged organisms. To determine whether these negative features of aging can be prevented by maintaining efficient autophagic activity until late in life, in this work we have corrected the CMA defect in aged rodents. We have generated a double transgenic mouse model in which the amount of the lysosomal receptor for CMA, previously shown to decrease in abundance with age, can be modulated. We have analyzed in this model the consequences of preventing the age-dependent decrease in receptor abundance in aged rodents at the cellular and organ levels. We show here that CMA activity is maintained until advanced ages if the decrease in the receptor abundance is prevented and that preservation of autophagic activity is associated with lower intracellular accumulation of damaged proteins, better ability to handle protein damage and improved organ function.

To summarize: In normal mice, a lysosomal protein required for autophagy gradually disappears with age, resulting in downregulation of autophagic activity. In the transgenics, levels of that protein can be artificially maintained, resulting in long-term maintenance of autophagy and a significant delay in functional deterioration of at least one organ (the liver).

The data strongly support a causative role for diminishing autophagy in hepatic aging; the transgenic model thus points the way toward a therapeutic intervention against one of aging’s root causes. How that would translate into practical terms is another matter: We don’t have the technology to alter the cellular genome in every cell of the body (nor is it clear we’d want to try: as an exercise, multiply [the rate at which your favorite hypothetical delivery method would mistarget an integration to an oncogene] by [the number of cells in the human body], and count the number of raging tumors that emerge). It might be faster, not to mention safer, if we could develop a small molecule capable of stimulating cells to maintain chaperone-mediated autophagy on their own.

It will be important to determine whether these results will hold true in other organs besides the liver. I’m particularly concerned about the brain, where previous experiments involving pharmaceutical upregulation of autophagy have had worrisome results. I would also love to know whether this method is effective at reversing, rather than preventing, age-related damage (e.g., in mice that have aged normally for some time before the transgene is activated).

On a final note: It seems intuitive that accumulating detritus will be bad for cells, but I think it would be fruitful to consider the mechanisms involved. What are the rate-limiting cellular functions that are the first to go when autophagy begins to fail? Another strategy, complementary to the one outlined in this paper, might be to boost the pathways that are the most sensitive to rising levels of junk, thereby making cells more tolerant to a given level of garbage. (In the end, of course, one would still have to reactivate autophagy in order to take out the trash, but simultaneously boosting stress tolerance might buy the cell some extra time.)

P.S.: This story was reported in the press a few weeks ago. Here’s a particularly choice piece of coverage from Down Under: Scientists stop the ageing process — holy smokes, we’re out of a job! Note the bizarre non sequitur stock photo.

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