Structural modeling and role of HAX-1 as a positive allosteric modulator of human serine protease HtrA2

Abstract

HAX-1, a multifunctional protein involved in cell proliferation, calcium homeostasis, and regulation of apoptosis, is a promising therapeutic target. It regulates apoptosis through multiple pathways, understanding of which is limited by the obscurity of its structural details and its intricate interaction with its cellular partners. Therefore, using computational modeling, biochemical, functional enzymology and spectroscopic tools, we predicted the structure of HAX-1 as well as delineated its interaction with one of it pro-apoptotic partner, HtrA2. In this study, three-dimensional structure of HAX-1 was predicted by threading and ab initio tools that were validated using limited proteolysis and fluorescence quenching studies. Our pull-down studies distinctly demonstrate that the interaction of HtrA2 with HAX-1 is directly through its protease domain and not via the conventional PDZ domain. Enzymology studies further depicted that HAX-1 acts as an allosteric activator of HtrA2. This ‘allosteric regulation’ offers promising opportunities for the specific control and functional modulation of a wide range of biological processes associated with HtrA2. Hence, this study for the first time dissects the structural architecture of HAX-1 and elucidates its role in PDZ-independent activation of HtrA2.

Purification, characterization and functional site prediction of the vaccinia-related kinase 2A small transmembrane domain

“…Overall, the thermal denaturation data demonstrated gain in stability of GST by ∼10°C in presence of the VTMD domain. Tertiary structure analysis by fluorescence spectroscopy Fluorescence emission spectra of GVTMD, VTMD and GST only samples in the absence and presence of chemical denaturant 6M urea were measured by a Fluorolog-3 spectrofluorometer (HORIBA Scientific, Edison, NJ, USA) using a quartz cell with 1 mm path-length cuvette at 25°C [31] . Emission data of 17.5 μM protein solutions in PBS buffer, pH 7.0 were recorded with 275 nm excitation followed by emission between 300-400 nm.…”

“…Fluorescence emission spectra of GVTMD, VTMD and GST only samples in the absence and presence of chemical denaturant 6M urea were measured by a Fluorolog-3 spectrofluorometer (HORIBA Scientific, Edison, NJ, USA) using a quartz cell with 1 mm path-length cuvette at 25°C [31] .…”

Unraveling the Dichotomy of Enigmatic Serine Protease HtrA2

“…This multitiered regulation of HtrA2 activation might be critical toward prevention of untimely proteolysis as well as accurately controlling its involvement in different pathophysiological pathways such as apoptosis, protein quality-control, cancer, arthritis, and neurodegeneration, where it cleaves a wide spectrum of substrates in different subcellular locations. This is substantiated by the identification and characterization of protein-protein interactions involving HtrA2 and its substrates such as Inhibitor of Apoptosis Proteins (IAPs), hematopoietic cell-specific protein-1-(HS1)-associated protein X-1 (Hax-1), Dual-specificity phosphatase-9 (DUSP-9), a gene associated with retinoic and interferon-induced mortality-19 protein (GRIM-19) and Phosphoprotein enriched in astrocytes-15 (Pea-15) (Chaganti et al, 2019;Acharya et al, 2020;Kummari et al, 2021) that unlike other HtrAs are interestingly not restricted to the C-terminal PDZ domains. The holistic enumeration of HtrA2's activation network has been vividly illustrated in Figure 2 and the mechanism is elaborated in the figure legend.…”

“…The binding of substrates to the hydrophobic YIGV groove allosterically activates the protease for substrate binding and subsequent catalysis. Furthermore, binding to mitochondrial substrates at the early apoptotic stage such as GRIM-19 and Hax-1 might be important toward attuning the mature protease for its proapoptotic functions before it enters the cytoplasm (Cilenti et al, 2004;Ma et al, 2007;Chaganti et al, 2019;Kummari et al, 2021) where it binds several antiapoptotic proteins including IAPs and death effector domain (DED) containing Pea-15 (Trencia et al, 2004). HtrA2 is also capable of inducing caspase-independent apoptosis via its serine protease activity by cleaving several critical cellular molecules such as cytoskeletal proteins (actin, α-/β-tubulin, and vimentin) that are important for upholding cellular integrity (Vande Walle et al, 2007).…”

Elucidating the role of GRIM-19 as a substrate and allosteric activator of pro-apoptotic serine protease HtrA2

“…HtrA2, with its basal activity is present in an inactive form in the mitochondria till it receives an apoptotic signal[51]. Initial activation of the protease might occur in the mitochondria in presence of its mitochondrial substrates such as Hax-1[52] and GRIM-19. Moreover, GRIM-19 that acts also as its activator might simultaneously facilitate attainment of required conformational changes for optimal activation of the protease.…”