Alzheimer’s disease (AD) pathogenesis involves complex, multifactorial changes to the brain proteome that conventional unfractionated analyses may obscure. Proteins frequently occupy multiple subcellular compartments as spatial proteoforms, yet the contribution of aberrant protein localization to AD pathogenesis remains poorly understood. To address this, we fractionated post-mortem human hippocampi from 13 AD and 14 non-AD individuals into four subcellular fractions and quantified 6,123 proteins by TMT-LC-MS. Although 75% of proteins were detected in more than one fraction, 78% of significant AD-associated alterations were restricted to a single fraction, demonstrating that subcellular localization is a primary determinant of disease vulnerability. Discordant abundance patterns between fractions revealed retromer complex mislocalization, nuclear transport dysfunction, and insoluble protein accumulation, with the endosomal-lysosomal and protein folding pathways most consistently perturbed. To examine how these perturbations evolve with disease progression, we applied the QUAD strategy to measure protein degradation in two fractions of APPswePS1delta9 mouse cortex at 2, 5, and 12 months. Degradation rates diverged between fractions and genotypes in an age-dependent manner, and cross-dataset comparison identified six proteins altered at the earliest pre-pathological timepoint, implicating vesicle transport and proteostasis disruption as initiating features of AD. These findings establish spatial proteoforms as essential units of pathogenic analysis and reveal disease-relevant signals invisible to bulk tissue approaches.
Daniel B. McClatchy, Natalie P. Turner, John R. Yates· bioRxiv· 0 citations
Although macrophages are important for controlling Group A Streptococcus (GAS) infection, we and others have demonstrated that GAS can persist in macrophages by perforating the phagolysosome using the pore-forming toxin streptolysin O (SLO). In this study, we identified lysosomal and bacterial proteins released into the cytosol as a consequence of phagosomal perforation.
We prepared cytosolic preparations from macrophages infected with either wild-type (WT) or SLO-deficient (α”SLO) bacteria and uninfected controls and verified lysosomal or bacterial proteins were present using IL-1β as a measure of intracellular pathogen detection. Proteomic analysis revealed distinct cytosolic protein profiles in both WT- and α”SLO-infected macrophages.
Bacterial M1 protein was detected only in the cytosol of WT-infected macrophages and corresponded with an IL-1β response, indicating SLO-mediated release of M1 protein from the phagosome, and providing a mechanism for cytosolic recognition of this virulence factor. Unexpectedly, cytosolic extracts of both WT- and α”SLO-infected macrophages contained all histone proteins, suggesting that nucleosomal complexes are released into the cytosol during GAS infection. We confirmed the presence of histones and the absence of contaminating nuclei in the cytosolic fraction by Western blot. DNA was not detected in the cytosol of GAS-infected cells, but histones were secreted into the extracellular medium. We are currently exploring the mechanism and purpose of this histone release.
Our data both confirms host cell detection of bacterial proteins after phagosomal perforation, as well as reveals the surprising profile of proteins altered during GAS infection.
NIH R15AI176429, AHA 17GRNT33410851, Occidental College
Microbial, Parasitic, and Fungal Immunology (MPF)
Cheryl Y. M. Okumura, A. Quezada, Kevin Lord et al.· Journal of Immunology· 0 citations