Aggregate Interactome Based on Protein Cross-linking Interfaces Predicts Drug Targets to Limit Aggregation in Neurodegenerative Diseases

Abstract

SummaryDiagnosis of neurodegenerative diseases hinges on “seed” proteins detected in disease-specific aggregates. These inclusions contain diverse constituents, adhering through aberrant interactions that our prior data indicate are nonrandom. To define preferential protein-protein contacts mediating aggregate coalescence, we created click-chemistry reagents that cross-link neighboring proteins within human, APPSw-driven, neuroblastoma-cell aggregates. These reagents incorporate a biotinyl group to efficiently recover linked tryptic-peptide pairs. Mass-spectroscopy outputs were screened for all possible peptide pairs in the aggregate proteome. These empirical linkages, ranked by abundance, implicate a protein-adherence network termed the “aggregate contactome.” Critical hubs and hub-hub interactions were assessed by RNAi-mediated rescue of chemotaxis in aging nematodes, and aggregation-driving properties were inferred by multivariate regression and neural-network approaches. Aspirin, while disrupting aggregation, greatly simplified the aggregate contactome. This approach, and the dynamic model of aggregate accrual it implies, reveals the architecture of insoluble-aggregate networks and may reveal targets susceptible to interventions to ameliorate protein-aggregation diseases.

Structural modeling of GSK3β implicates the inactive (DFG-out) conformation as the target bound by TDZD analogs

“…For experiments with drug treatment cells were plated at 6000 cells per well in a 96-well tissue culture treated plate (Costar). Drug-treated cells and controls were stained with thioflavin-T as previously described 41 , 44 , 45 . Cells were fixed after 48-h drug exposure, and incubated in phosphate-buffered saline containing 0.1% (w/v) thioflavin-T dye, and 4′,6-diamidino-2-phenylindole (DAPI).…”

“Protein aggregates” contain RNA and DNA, entrapped by misfolded proteins but largely rescued by slowing translational elongation

“…We began with total, sarkosyl‐insoluble aggregates isolated from SY5Y‐APP Sw human neuroblastoma cells (Ayyadevara et al, 2017), a model of familial Alzheimer's disease (fAD). This work revealed a complex, non‐random structure of aggregates in which megahubs (very‐high‐connectivity hubs with ≥100 partners) and hub connectors (low‐connectivity proteins linking large hubs) contribute functionally to the assembly of large aggregates (Balasubramaniam et al, 2019). We noted marked enrichment among megahubs for large structural proteins such as titin, ankyrins 1 – 3, nesprins 1 – 3, MAP1A, and other neurofilament proteins, purely as a consequence of their size.…”

“…aggregation, Alzheimer's disease, apolipoprotein E, beta amyloid, cotranslational misfolding, DNA, endogenous viruses, functional annotation, gene ontology, neurodegeneration, nucleic acid sequence, nucleic acids, protein aggregates, proteomics, retrotransposons, RNA F I G U R E 1 The "aggregate contactome" of proteins isolated from SY5Y-APP Sw human neuroblastoma cells, an in vitro model of familial AD. The contactome was generated from proteomic data for cross-linked peptide pairs in sarkosyl-insoluble aggregates, using a modified version of X-link Identifier (Balasubramaniam et al, 2019;Du et al, 2011), requiring ≥10 spectral hits per protein observed in at least 2 of 3 replicate crosslinking experiments. Hits were normalized to hub length (amino acids in the most abundant isoform).…”

From fuzziness to precision medicine: on the rapidly evolving proteomics with implications in mitochondrial connectivity to rare human disease

“…For example, Aβ 42 and tau are persistently found in AD aggregates as an AD pathological hallmark. This suggests that seed constituents likely coordinate common and may also mediate preferential PPIs within aggregate interfaces in different patients, which can be traced to discover individual-specific biomarkers for blockage using aggregate dispersal therapies ( Balasubramaniam et al., 2019 ). Anti-aggregate therapeutics aim to diffuse these pathological “protein lumps” in AD and other neurological diseases.…”

“…This is followed by chemical cross-linking, tryptic digest, and LC-MS/MS analysis to detect cross-linked peptide spectra. This method has successfully revealed 506 contactome protein hubs that are involved in >7,000 interactions, with ~6,000 interactions deemed specific to the disease cell line model as opposed to the non-mutant counterpart ( Balasubramaniam et al., 2019 ), which highlights the complexity in protein aggregate compositions in AD. For illustration, we used molecular docking to predict the interaction interface between the reported interacting proteins SRSF6, Tau, and APP for the subsequent development of aggregate dispersal drugs ( Figure 3 C).…”