Submission
| Title: |
Thermal proteome profiling of lymphoblastic cell lines from patients with Diamond-Blackfan Anemia reveals thermal stability differences in ubiquitin-associated pathways |
| Authors: |
Wijeratne, H.R. Sagara, Department of Biochemistry & Molecular Biology, Indiana University School of Medicine; Avery Runnebohm, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine; Sarah A.Peck-Justice, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine; Neil A. McCracken, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine; Emma H. Doud, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine; Jungsu Kim, Department of Medical and Molecular Genetics, Indiana University School of Medicine; Ugo Ramenghi, Department of Public Health and Pediatric Sciences, University of Torino; Anna Aspesi, Department of Health Sciences, Università del Piemonte Orientale; Amber L. Mosley, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine |
Abstract
Background/Significance/Rationale: An increasing number of genetic variants are being associated with rare diseases due to the advent of high-throughput sequencing. However, it is difficult to characterize molecular mechanisms of the variant in the pathology in a high-throughput manner. Diamond-Blackfan Anemia (DBA), a rare bone marrow disorder that prevents the development of red blood cells, is caused primarily by haploinsufficiency of one of the ribosomal protein subunits. How these different proteins result in the same disease is unknown. Here we are using a mass-spectrometry-based technique called thermal proteome profiling (TPP) to determine changes in thermal stability of the proteome as a consequence of expression of DBA-associated variants.
Methods: Lymphoblastic cell lines were derived from patients with pathological variants in RPS19, RPL5, RPL35A, and RPS24 and healthy donor controls. TMT-based global proteomics was performed in parallel to TPP. Differential abundance was determined using MSStatsTMT with an adjusted p-value ≤ 0.05. TPP was performed for eight temperature treatments. Protein melt curves/temperatures were determined using R package Inflect-SSP. Statistically significant thermal shifts were set at a cutoff of p ≤ 0.05.
Results/Findings: Analysis of the DBA cell lines revealed 236 proteins with significant thermal shifts. Enrichment analysis of these proteins using gprofiler2 found terms involving ribosomal, recapitulating the known biology of the pathogenesis of DBA. We also discovered proteins suggesting a role of the ubiquitin-proteasome system (UPS) in DBA. Proteins associated with microcephaly, a clinical feature of DBA that affects a small percentage of patients, were also enriched. These findings reveal novel insights into the cellular dysfunction associated with DBA-causing protein variants including precise targets within the extensive UPS – with specific E3 ligases and regulatory proteins identified with thermal stability changes.
Conclusions/Discussion: TPP show a role of the ubiquitin-proteasome system in the Diamond-Blackfan Anemia disease process.
Translational/Human Health Impact: Various types of protein functional variants that lead to rare diseases can be profiled effectively by thermal proteome profiling to give new molecular insights into disease processes.
