Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

<div><p>Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration.</p></div

Quantified proteins are expressed in all major cellular components.

<p>(<b>A</b>) A Venn diagram showing that more than 50% of the 6070 quantified proteins (protein isoforms encoded by the same gene are merged) are shared among the soluble and insoluble fractions of both the CG and CL groups. (<b>B</b>) The percentage of quantified proteins among all annotated proteins for each GO term of the cellular components category. (<b>C</b>) For each cellular component GO term listed in the middle, the distribution (%) of these proteins among all quantified proteins is shown on the left, and the number of proteins identified and quantified only in the soluble fraction, or only in the insoluble fraction, or in both, is shown on the right. Note that no proteins of the respiratory chain were uniquely identified and quantified in the soluble fractions (red arrow).</p

Sleep deprivation affects proteins that are associated with multiple subcellular components and biological processes, especially mitochondrial proteins that functioning in energy and small molecule metabolism.

<p>(<b>A</b>) Enrichment of GO terms in the 'cellular components' category among the up- and down-regulated proteins in the insoluble and soluble fractions of all three biological replicates from CG and CL. As shown in the heat map, the proteins up-regulated by sleep deprivation are significantly and consistently enriched for GO terms related to mitochondria, especially the mitochondrial inner membrane. (<b>B</b>) Enrichment of GO terms in the 'biological processes' category among the up- and down-regulated proteins. Up-regulated proteins from the insoluble fractions are significantly and consistently enriched for GO terms related to energy and small molecule metabolism, while those from the soluble fractions are enriched for GO terms of transport function. In contrast, proteins down-regulated by sleep deprivation were miscellaneous in their distribution, and were less consistent. The color scale of the <i>p</i> values for the significance of enrichment is the same as the scale in (A).</p

Proteins up-regulated by sleep deprivation are linked to neurodegeneration diseases.

<p>According to the annotations of the KEGG Disease and the Human Phenotype Ontology databases, the 140 up-regulated proteins in the CG or CL groups, or both, were enriched for proteins associated with human diseases. The bars show the percentage (left) and–ln(<i>p</i> value) (right) of the enriched annotation categories (*, p<0.05, -ln(p value) >3). Up-regulated proteins are significantly enriched in Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease.</p

Proteins showing abundance changes after sleep deprivation by both GSD and LSD.

<p>Proteins showing abundance changes after sleep deprivation by both GSD and LSD.</p

Sleep deprivation resulted in the up-regulation of proteins involved in cellular respiration.

<p>(<b>A</b>) Venn diagrams showing the distribution of significantly up-regulated proteins in the CG and CL groups. The overlap of 22 overlapped proteins is detailed in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163500#pone.0163500.t001" target="_blank">Table 1</a></i>. (<b>B</b>) All of the complexes in the electron transport chain were influenced by sleep deprivation. Complex I-III had only up-regulated subunits, but Complex IV and V had both up- and down-regulated subunits. Dysregulated proteins are listed in the coloured table. Up-regulated proteins are in red boxes; down-regulated proteins are in green. Double arrows represent changes common to both the CG and CL sample groups. (<b>C</b>) Proteins involved in small molecule metabolism were collectively up-regulated. Metabolic compounds are framed in orange boxes, and connected with one another with solid or dashed lines, which indicate direct or indirect conversions, respectively. Enzymes that are found up-regulated from the CG or the CL groups are enclosed in parentheses or square brackets, respectively, or presented with bold font if increased in both groups. (<b>D</b>) After being synthesized in the cytosol, preproteins enter the mitochondria through the translocase of the outer membrane (TOM) followed by the translocase of the inner membrane (TIM). TIMM9 was up-regulated in both SD groups. During its transit, polypeptide can be bound and stabilized by mtHsp70 (encoded by Hspa9) with the help of Grpel1. Next, the preprotein is handed over to the complex composed of Hsp60 (encoded by Hspd1) and Hsp10 (encoded by Hspe1) and is further assisted with folding. Upon reaching their native states, mature mitochondrial proteins are sorted to their final destinations. Proteins damaged either by ROS attack or misfolding are repaired by entering refolding cycles or are degraded with the assistance of these chaperones. Chaperones that are found up-regulated from the CG or the CL groups are enclosed in parentheses or square brackets, respectively, or presented in bold font if found to be increased in both groups.</p

Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16^XPF and Inhibiting Dna2

<div><p>Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16^XPF-Swi10^ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3′ endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5′ endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases.</p></div

Pxd1 acts with Rad16-Swi10 in the IR response.

<p>(A) The DNA damage sensitivity of the indicated strains was examined using a spot assay. <i>pxd1Δ</i>, but not <i>saw1Δ</i>, cells exhibited mild IR sensitivity. The IR sensitivity of <i>rad16Δ</i> and <i>swi10Δ</i> cells was stronger than that of NER-defective <i>rhp14Δ</i> cells, suggesting a role of Rad16-Swi10 in non-NER repair. (B) Deletion of <i>saw1</i> did not alter the DNA damage sensitivity of <i>pxd1Δ</i>, <i>rad16Δ</i>, or their double mutant. (C) For the IR sensitivity phenotype, <i>rad16Δ</i> is epistatic to <i>rhp14Δ</i> and <i>pxd1Δ</i>. The double mutant <i>rhp14Δ pxd1Δ</i> was more sensitive than <i>rhp14Δ</i> or <i>pxd1Δ</i> and phenocopied <i>rad16Δ</i>, suggesting that Pxd1 acts with Rad16 in the non-NER repair process.</p

Pxd1 interacts with Rad16-Swi10 and Dna2-Cdc24.

<p>(A) Proteins that co-purified with Saw1. Saw1 tagged with a YFP-FLAG-His₆ (YFH) tag was purified using anti-YFP beads and analyzed by mass spectrometry. (B) Proteins that co-purified with Pxd1. (C) Pxd1 interacts with Rad16-Swi10 and Dna2-Cdc24 through its middle and C-terminal regions, respectively. Full-length (FL) and truncated Pxd1 proteins fused with the TAP tag were immunoprecipitated using IgG beads. The co-immunoprecipitation of Myc-tagged Rad16 or Cdc24 was analyzed by immunoblotting. The N-terminal region of Pxd1 is prone to be cleaved off by proteolysis. The bottom panel depicts the Pxd1 truncations and summarizes the co-immunoprecipitation results. (D) Pxd1 is required for the association between Rad16 and Cdc24. Cdc24-Myc was co-immunoprecipitated with Rad16-TAP in the wild-type background, but not in the <i>pxd1Δ</i> background. (E) Pxd1 is required for the association between Saw1 and Cdc24. Cdc24-Myc was co-immunoprecipitated with Saw1-TAP in the wild-type background, but not in the <i>pxd1Δ</i> background. (F) Schematic of the inferred organization of the PXD complex.</p

Pxd1 acts with Rad16-Swi10 in mating-type switching and the removal of covalent Top1-DNA adducts.

<p>(A) <i>rad16Δ</i> and <i>pxd1Δ</i>, but not <i>saw1Δ</i>, cells are defective in mating-type switching. The <i>h⁹⁰</i> strains with the indicated genotypes were spread onto malt extract (ME) plates, and single colonies were allowed to form before they were stained with iodine vapor. (B) Increased Rad52 accumulation at the mating type locus was observed in <i>rad16Δ h⁻</i> and <i>pxd1Δ h⁻</i> cells. Strand-specific Rad52 ChIP-seq was performed as described <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001946#pbio.1001946-Zhou1" target="_blank">[43]</a>. (C) The middle region of Pxd1 is required for its mating-type switching function. Mating-type switching assay was performed as in (A). (D) <i>tdp1Δ</i> and <i>pxd1Δ</i> are synthetic lethal/sick in a Top1-dependent manner. Representative tetrads from a cross between a <i>pxd1Δ</i> strain and a <i>top1Δ tdp1Δ</i> strain are shown. (E) The middle region of Pxd1 is required to rescue the synthetic lethality/sickness of the <i>tdp1Δ pxd1Δ</i> cells. Shown are representative tetrads from crosses between <i>pxd1Δ</i> strains transformed with a plasmid expressing full-length or middle-region-deleted Pxd1 and a <i>top1Δ tdp1Δ</i> strain. The plasmid was integrated at the <i>pxd1</i> locus.</p