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Extremely Long-Lived Nuclear Pore Proteins in the Rat Brain

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Science  24 Feb 2012:
Vol. 335, Issue 6071, pp. 942
DOI: 10.1126/science.1217421

Abstract

To combat the functional decline of the proteome, cells use the process of protein turnover to replace potentially impaired polypeptides with new functional copies. We found that extremely long-lived proteins (ELLPs) did not turn over in postmitotic cells of the rat central nervous system. These ELLPs were associated with chromatin and the nuclear pore complex, the central transport channels that mediate all molecular trafficking in and out of the nucleus. The longevity of these proteins would be expected to expose them to potentially harmful metabolites, putting them at risk of accumulating damage over extended periods of time. Thus, it is possible that failure to maintain proper levels and functional integrity of ELLPs in nonproliferative cells might contribute to age-related deterioration in cell and tissue function.

Functional deterioration and accumulation of damage to the proteome is largely repaired through protein turnover where potentially impaired polypeptides are replaced with new, functional copies. These turnover mechanisms are particularly important in postmitotic cells such as neurons, because they cannot dilute potentially toxic species through cell division. Nearly all proteins within the human proteome are recycled in less than a few days (1, 2). However, a few extremely long-lived proteins (ELLPs) with half-lives on the order of months have been identified (3, 4), including myelin basic protein (MBP). A subset of nuclear pore complex (NPC) proteins, which form transport channels responsible for mediating nuclear trafficking (5), are present but no longer expressed in differentiated cells (6). Thus, at least a subset of nucleoporins (Nups) are not, or are only very slowly, replaced during adulthood. However, because worms have a life span of a few weeks, it remains unclear whether NPC components remain incorporated in the nuclear membrane over years, particularly in the central nervous system of mammals, which contain nondividing cells that are as old as the organism itself (7).

To explore this question, we performed pulse chase labeling of whole rats with the stable isotope 15N followed by mass spectrometry to monitor global protein turnover on a time scale of years (the average life span of a lab rat is 2 years). Two female rats and their progeny were fed a 15N-enriched algal cell diet, and at 6 weeks all progeny rats were switched to a 14N diet. Fully 15N labeled rats were immediately killed and their tissues harvested. Nuclei from liver, an organ that turns over within 4 to 6 months, and brain were purified, digested with trypsin, and analyzed by MudPIT (multidimensional protein identification technology) LCLC-MS/MS (multidimensional liquid chromatography–tandem mass spectrometry). At time = 0, we calculated 15N isotopic protein labeling efficiency of >98% and identified more than 3400 fully 15N proteins (20,754 peptides) and only 9 14N proteins (14 peptides). Two additional animals were killed at 6 and 12 months, and 15N/14N ratios were determined for more than 3500 unique proteins. Only seven heavy (15N) proteins (11 peptides) were found in the liver after 6 months, consistent with the relatively rapid turnover of hepatocytes. In contrast, the brain contained a large number of heavy peptides (92 peptides) even after 12 months (Fig. 1, A and B, and fig. S1E). These peptides corresponded to 25 proteins and included MBP and histones, the latter having reported half-lives of ~220 days in mouse brain (8), confirming the validity of our approach. All the other heavy proteins identified were components of the two essential core modules of the NPC, the pentameric Nup205 complex and the nonameric Nup107-160 complex (6) (Fig. 1C and table S1). This represents an essential intracellular protein machine with protein components in excess of a year in age.

Fig. 1

Identification of NUPs and histones as ELLPs in mammalian brain. (A) MS1 scans [indicated mass/charge (m/z) ranges] at 0 and 6 months. Distinct peptides for indicated proteins; red indicates 15N peptide peaks; black, 14N; gray, other peptides. Asterisk peaks were successfully identified by MS/MS. (B) Distribution of 15N MS/MS spectral counts (485) grouped as proteins (25) from 12-month brain nuclei and 6-month liver nuclei (inset). (C) Relative MS1 peak quantitation for each Nup with heavy peptide hits indicated as 15N/14N ratios when possible.

Detailed analysis of 15N spectral counts and 15N/14N MS1 ratios revealed that, in contrast to the stable scaffold, the peripheral Nups and components of the central transport channel were devoid of heavy peptides, suggesting they were completely replaced after 6 months (Fig. 1C). Thus, unlike other large protein complexes in which all components have similar turnover values (1, 2), the individual components of NPCs have very different lifetimes. This supports the idea that NPCs are built to last the entire life span of the cell and are not completely removed and assembled anew in postmitotic cells. Rather, NPC maintenance in nondividing cells relies on the non- or extremely slow exchange of scaffold and rapid replacement of peripheral Nups.

A lack of protein turnover exposes the proteome to an increased risk of aberrant chemical modifications and oxidative damage during aging. Indeed, healthy rats exhibit age-dependent decline of NPC function (6). Our results suggest that ELLPs represent a diverse class of proteins that regulate essential cellular functions and could be linked directly to the decline of the aging proteome.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1217421/DC1

Materials and Methods

Fig. S1

Table S1

References (919)

References and Notes

  1. Acknowledgments: We are supported by the Hewitt Foundation (B.H.T.); the Ellison Medical and Glenn foundations (M.W.H.); National Institute of Aging fellowship F32AG039127 (J.N.S.); and NIH grants P41 RR011823, P01 AG031097, HHSN268201000035C, and R01 MH067880 (J.R.Y. and J.N.S.) and P30 CA014195 (M.W.H.). The RAW files and parameter files are publicly available at http://fields.scripps.edu/published/ellnpp2011/.
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