Shift of the insoluble content of the proteome in the aging mouse brain

Abstract

During aging, the cellular response to unfolded proteins is believed to decline, resulting in diminished proteostasis. In model organisms, such as Caenorhabditis elegans, proteostatic decline with age has been linked to proteome solubility shifts and the onset of protein aggregation. However, this correlation has not been extensively characterized in aging mammals. To uncover age-dependent changes in the insoluble portion of a mammalian proteome, we analyzed the detergent-insoluble fraction of mouse brain tissue by mass spectrometry. We identified a group of 171 proteins, including the small heat shock protein α-crystallin, that become enriched in the detergent-insoluble fraction obtained from old mice. To enhance our ability to detect features associated with proteins in that fraction, we complemented our data with a meta-analysis of studies reporting the detergent-insoluble proteins in various mouse models of aging and neurodegeneration. Strikingly, insoluble proteins from young and old mice are distinct in several features in our study and across the collected literature data. In younger mice, proteins are more likely to be disordered, part of membraneless organelles, and involved in RNA binding. These traits become less prominent with age, as an increased number of structured proteins enter the pellet fraction. This analysis suggests that age-related changes to proteome organization lead a group of proteins with specific features to become detergent-insoluble. Importantly, these features are not consistent with those associated with proteins driving membraneless organelle formation. We see no evidence in our system of a general increase of condensate proteins in the detergent-insoluble fraction with age.

Analysis of protein levels and solubility in distinct brain regions reveals several elements of the protein homeostasis network that are impacted by aging

“…12 Using the same 14 features that were identified in our previous study, the two populations of detergent-insoluble proteins derived from either the hippocampus or the cortex are markedly separated based on age in a PCA diagram with 72.3% of the variation captured in dimension 1 (Figure 3A). 12 The insoluble proteins enriched in the old mice are correlated with features such as β-sheet content, percentage of sequence in Pfam domains and hydrophobicity. Similarly, detergent-insoluble proteins in aged mice are significantly depleted for intrinsic disorder and have more of their sequence in Pfam domains when compared to the whole mouse proteome (Figure 3B).…”

“…During this time, they were fed a standard diet (Teklad X2920, Envigo, USA) Mice were sacrificed and processed to obtain whole brain regions and sagittal hemisphere sections as previously described. 12 Briefly, mice were anesthetized using isoflurane gas followed by and perfusion through the left ventricle with 20mL of 1xPBS containing 1x HALT protease and phosphatase inhibitors. For the lysate-based experiments (MS and FTA), the cortex and hippocampus were collected and snap frozen in liquid nitrogen.…”

“…MS analysis was performed as previously described. 12 Detergent solubility samples were run on a Q exactive HF mass spectrometer. Online fractionation was performed with an Easy-nLC 1200 liquid chromatography system (Thermo Scientific) configured with a 20µl sample loop, 30 µm ID steel emitter and a 50 cm µPACTM analytical column (Pharmafluidics) with trap column heated to 50°C.…”

“…11 We previously assessed by mass spectrometry (MS) the presence of triton-insoluble proteins in mouse brains and showed that a small subset of the proteome becomes more insoluble upon aging. 12 These age-associated insoluble proteins tend to be more structured and depleted for intrinsic disordered. Proteins with a similar physicochemical "signature" are found to be insoluble in several other proteomics mouse studies of different ND models, but only when older mice are assessed.…”

Capture of the Mouse Organ Membrane Proteome Specificity in Peptidisc Libraries

“…The precise knowledge of these organ-specific proteins, whose expression is significantly elevated in a given organ, holds great medical significance. For instance, several clinical diagnostics rely upon organ-specific protein biomarkers to monitor pathological states and disease progression, whereas the development of therapeutics critically depends on understanding organ specificity to prevent cross-reactivity. , This knowledge is especially important at the cell surface since about two-thirds of the druggable proteome comprises integral membrane proteins. , Perturbation of the membrane proteome is also linked to neurodegenerative diseases, cancer, and disorders like hypertension and muscular dystrophy. − Genomic and transcriptomic technologies have undeniably proven their worth by shedding light on organ protein expression patterns. − Still, to achieve a comprehensive understanding of organ biology at a molecular level, a direct estimate of its protein content is imperative. − …”