Cellular accumulation of misfolded proteins is a hallmark of ageing. In young cells, the proteostasis network limits toxicity by activating one or more systems for misfolded protein clearance. We focus on how these clearance systems are integrated within the network to maintain proteome health during youth, and how their dis-integration contributes to cellular senescence—another ageing hallmark with strong links to chronic inflammation and organismal frailty.
We currently use two evolutionarily distant cell types—budding yeast and primary human fibroblasts—to identify common, conserved lines of communication between different clearance systems of the proteostasis network, and how these get re-wired during replicative ageing (yeast) and senescence (mammals). Our lab employs multi-disciplinary approaches such as super-resolution imaging, flow cytometry, and mass-spectrometry-based proteomics to measure proteostasis capacity and senescence phenotypes as quantitatively and robustly as possible. As proteostasis modulators hold therapeutic promise in ageing-associated pathologies—with renewed interest in ‘senolytics’ specifically targeting senescent cells—we hope to drive fundamental discoveries that have a direct impact on promoting lifelong health.
We use a multi-disciplinary approach (left panel), using high-resolution imaging, flow cytometry, and mass spectrometry-based proteomics, to probe the relationship between loss of proteostasis and onset of senescence—two of the hallmarks of ageing (middle—adapted from Lopéz-Otín et al., 2013). Our current focus is on the interplay between different protein clearance systems in young vs. senescent cells (right).
Check out Rahul’s talk from Methuselah Health UK’s Conference on Why We Age (2020), entitled “Why we turnover our proteins (and how it gets done)"
Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads
Harvey E. Johnston, Kranthikumar Yadav, Joanna M. Kirkpatrick, George S. Biggs, David Oxley, Holger B. Kramer, Rahul S. Samant
Native size exclusion chromatography-based mass spectrometry (SEC-MS) identifies novel components of the Heat Shock Protein 90-dependent proteome
Rahul S. Samant, Silvia Batista, Mark Larance, Bugra Ozer, Christopher I. Milton, Isabell Bludau, Laura Biggins, Simon Andrews, Alexia Hervieu, View Harvey E. Johnston, Bissan Al-Lazikhani, Angus I. Lamond, Paul A. Clarke, Paul Workman
The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.
Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetism─with optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1-5000 μg preparations and improved reproducibility (median protein 0.99 (SP4) 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffers─all confirming equivalent or improved proteome characterization SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications─all while retaining the speed and compatibility of SP3.
The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.