Globally Analyzing and Dissecting Apoptosis
Examples of our research and methods include
Creating ligases, altering proenzymes
Department scientists have developed several protein engineering approaches toward the dissection of apoptosis:
- Redesigning a bacterial enzyme (from subtilisin to subtiligase) so it labels the exposed alpha-amines (N-termini) of cleaved proteins to globally identify substrates, caspase-like cleavage sites, and relative reaction rates (catalytic efficiencies), as well as common cross-species caspase targets (evolutionarily conserved, thus hypothetically crucial) via mass spectrometry
- Engineering inactive caspase precursors (proenzymes) so that each executioner caspase can be selectively activated in order to dissect their apoptotic contributions and targets
Our applications of such protein engineering include
Determining caspase specificity in response to chemotherapy
Using subtiligase and mass spectrometry to identify specific caspase-like cleavage sites in hundreds of different substrates in human cells in which apoptosis was induced by chemotherapy (doubling the number of then-known cleavage sites in human targets). Global analysis found that the identified caspase substrates disproportionately interact, suggesting the enzymes target protein complexes and networks to bring about apoptosis.
Tracking prioritization of apoptotic targets
Quantitatively tracking the appearance of hundreds of cleaved substrates induced by apoptotic caspases as a function of time, in order to determine hundreds of catalytic efficiencies in parallel and thus the prioritization of the enzymes’ targets. Findings indicated a sequential process with more rapid targeting of specific cellular processes.
Parsing roles of executioner caspase isoforms
Engineering proenzymes of apoptotic caspases with activating cleavage sites targeted by a non-human protease (tobacco etch virus) under small molecule control. This allowed researchers to dissect individual isoform roles, including which caspases’ activation was alone sufficient to induce apoptosis. The approach, dubbed SNIPer (single nick in proteome), also suggested that executioner caspases are substrates of the proteasome (protein complexes that destroy unwanted proteins inside cells), which is, in turn, targeted by the activated cell-demolishing enzymes. The latter result suggested beneficial clinical synergies between proteasome inhibitors (an existing cancer therapy) and proapoptotic drugs.
Selectively ablating neurons in brain study
Using the targeted viral delivery of proenzymes of apoptotic caspases engineered for activation by heterologous proteases (see above) to selectively ablate a tiny sub-set of neurons in the brains of mice (about 2,000 out of 100 million). This collaboration with UCSF neuroanatomists parsed the roles of specific sexually dimorphic neurons (those in the ventromedial hypothalamus expressing progesterone receptor proteins), demonstrating that they govern mating behaviors in both sexes as well as aggression in males.