This article was co-authored by Dhwani Rupani, Dhruvika Chawalla and Vidhi Khanna

The mechanism of activation of the pro-apoptotic protein HtrA2 has been elucidated by researchers at the Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai. HtrA2 (high temperature requirement protease A2), a unique mitochondrial pro-apoptotic protein, conserved from prokaryotes to humans is involved in functions such as apoptosis, protein quality control, unfolded protein response, cell growth and metabolism of amyloid precursor proteins. It is also implicated in several diseases including arthritis, neurodegenerative disorders and cancer making it an important therapeutic target. The x‑ray structure of HtrA2 could not explain clearly how crosstalk and plasticity of all domains, loops and linkers make it a fully functional molecule. As Dr. Kakoli Bose puts it, ​“Something triggers its activation and we wanted to know what.” Thus began a two year long research that has finally led to this.

HtrA2 has a trimeric pyramidal structure with short N‑terminal region holding the oligomer together through intermolecular hydrophobic and van der waals interactions, thus playing a role in the stability of the trimeric ensemble. The core consists of three serine protease domains (SPDs) surrounded by three C‑terminal PDZ domains restricting the entry of the substrate to the SPD active site. SPD is connected to PDZ through a flexible linker and it is hypothesized that PDZ-protease dynamics and their relative orientation modulate HtrA2 activity and hence functions. ​“It is the intermolecular interaction and not the intramolecular interaction that is essential here” quips Dr. Bose. She further adds that, activation of HtrA2 occurs allosterically, where the signal is relayed via a distal non-canonical substrate binding site i.e. the PDZ domain moves towards the adjacent SPD domain resulting in the formation of an oxyanion hole, or simply put ​‘the catalytic site’. It is these structural reorganizations and conformational dynamics that lead through HtrA2 activation and confirm the need for a trimeric structure rather than a monomeric one where the catalytic site would not be appropriately formed.

FRET (Förster resonance energy transfer) studies were conducted by Lalith K. Chaganti, Ph.D. student in Dr. Bose’s lab, to determine the intramolecular PDZ-SPD interaction and the dynamics of the SPD-PDZ interface. It was observed that in case of HtrA2, the energy transfer efficiency was much higher and the distance between PDZ and SPD was lesser as compared to HtrA2 mutant or the monomeric form, thus confirming their hypothesis. A model was propounded where, as a function of temperature, the interface movement occurs such that the PDZ moves away from SPD in a V‑shaped trajectory thus bringing the two FRET pairs in vicinity of each other. This model might also hold good for the activation of HtrA2 via substrate binding to PDZ. The team also performed studies using enzymology, in which FITC labeled b‑Casein was used as a substrate to check the activity of the enzyme via fluorescence studies. Kinetic parameters like K_m, V_max, etc were also quantitatively studied to correlate the observations with substrate binding and catalysis. Additionally, they also performed far UV-CD studies to understand the effect of deletions and mutations on HtrA2 secondary structure and stability. These studies highlight importance of N‑terminal region, oligomerization and intricate intermolecular PDZ-protease interaction in proper active-site formation, enzyme-substrate complex stabilization and hence HtrA2 functions. The fact that this study involved a number of advanced research techniques rather than just one, definitely gives it an edge over the others. It has experimental data such that one experiment backs the other. Next in line, Bose et al. are looking at HtrA2 homologues, specific substrates and also inhibitors, apart from HtrA2 they are also working on another human papillomavirus protein E2.

Ask Dr. Bose, why she chose this particular protein for her research and she elaborates that she wanted to work on a pro-apoptotic molecule with a complex structure-function relationship that is implicated in cancer. With HtrA2, that fulfills all these criteria, she seemed to have found her match. ​“More than anything else, HtrA2 acts via non-classical apoptotic pathways and has an extremely intriguing structure which pushed me to pursue this”, she says. HtrA2 has been implicated in a number of cancers, namely breast and prostate cancer; making it a promising therapeutic target. If the intricacies of how apoptotic pathways via HtrA2 are shut-down in case of cancer, its inhibitors can be identified, the re-establishment of these pathways will not be far away. This could provide a disease targeted rather than a symptom targeted solution to millions of patients suffering from cancer all over the world.