Elucidating Proteoform Dynamics Underlying the Senescence Associated Secretory Phenotype

Primary diploid cells exit the cell
cycle in response to exogenous
stress or oncogene activation through a process known as cellular
senescence. This cell-autonomous tumor-suppressive mechanism is also
a major mechanism operative in organismal aging. To date, temporal
aspects of senescence remain understudied. Therefore, we use quantitative
proteomics to investigate changes following forced HRASG12V expression and induction of senescence across 1 week in normal diploid
fibroblasts. We demonstrate that global intracellular proteomic changes
correlate with the emergence of the senescence-associated secretory
phenotype and the switch to robust cell cycle exit. The senescence
secretome reinforces cell cycle exit, yet is largely detrimental to
tissue homeostasis. Previous studies of secretomes rely on ELISA,
bottom-up proteomics or RNA-seq. To date, no study to date has examined
the proteoform complexity of secretomes to elucidate isoform-specific,
post-translational modifications or regulated cleavage of signal peptides.
Therefore, we use a quantitative top-down proteomics approach to define
the molecular complexity of secreted proteins <30 kDa. We identify
multiple forms of immune regulators with known activities and affinities
such as distinct forms of interleukin-8, as well as GROα and
HMGA1, and temporally resolve secreted proteoform dynamics. Together,
our work demonstrates the complexity of the secretome past individual
protein accessions and provides motivation for further proteoform-resolved
measurements of the secretome.