Globally Analyzing and Dissecting Apoptosis

There are four research areas in the Department of Pharmaceutical Chemistry. Globally analyzing and dissecting apoptosis is a research challenge within protein and cellular engineering.

The challenge

Apoptosis is the programmed cellular self-destruction vital to eliminating unneeded, infected, or tumorigenic cells. It fails to occur in cancers and can also occur too much, as in neurodegenerative diseases.

The highly regulated process involves various signaling cascades that eventually activate several members of a cysteine protease family, apoptotic caspases (and ultimately, the subset of “executioner” caspases-3, and -7), which spur cell demolition by cleaving more than 1,400 different proteins.

Determining which targets—including cellular machinery and network nodes—are key to apoptosis in different cell types would identify new and prime targets for anti-cancer drugs that are “downstream” in fundamental biological pathways and thus less vulnerable to resistant mutations.

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.

diagram showing endogenous apoptosis pathways and engineered caspase activation

The SNIPer Approach

  • Enlargement: A previously developed split form of a protease from the tobacco etch virus (TEV) is designed so that it can combine and activate only in the presence of a small molecule (blue hexagon)—the drug rapamycin.
  • Left box: Normally apoptotic caspases exist as inactive proenzymes, until external signaling molecules cause their proteolytic activation by upstream enzymes, including other caspases. (Here procaspase-8 and -9 are activated; they then activate the executioner caspase-3.)
  • Right box: In the SNIPer approach, executioner caspase isoforms (here procaspase-3) are engineered to include sequences recognized by TEV protease (which has no natural human targets). Activated by rapamycin, TEV protease cleaves / activates specific procaspases, thus revealing their contributions to apoptosis; e.g., caspase-3 targets cell proteasomes, protein complexes that act as brakes on apoptosis.

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.