Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway

Abstract

High-temperature requirement protease A4 (HtrA4) is a secretary serine protease whose expression is up-regulated in pre-eclampsia (PE) and hence is a possible biomarker of PE. It has also been altered in cancers such as glioblastoma, breast carcinoma, and prostate cancer making it an emerging therapeutic target. Among the human HtrAs, HtrA4 is the least characterized protease pertaining to both structure and its functions. Although the members of human HtrA family share a significant structural and functional conservation, subtle structural changes have been associated with certain distinct functional requirements. Therefore, intricate dissection of HtrA4 structural and functional properties becomes imperative to understand its role in various biological and pathophysiological processes. Here, using inter-disciplinary approaches including in silico, biochemical and biophysical studies, we have done a domain-wise dissection of HtrA4 to delineate the roles of the domains in regulating oligomerization, stability, protease activity, and specificity. Our findings distinctly demonstrate the importance of the N-terminal region in oligomerization, stability and hence the formation of a functional enzyme. In silico structural comparison of HtrA4 with other human HtrAs, enzymology studies and cleavage assays with X-linked inhibitor of apoptosis protein (XIAP) show overall structural conservation and allosteric mode of protease activation, which suggest functional redundancy within this protease family. However, significantly lower protease activity as compared with HtrA2 indicates an additional mode of regulation of the protease activity in the cellular milieu. Overall, these studies provide first-hand information on HtrA4 and its interaction with antiapoptotic XIAP thus implicating its involvement in the apoptotic pathway.

A distinct concerted mechanism of structural dynamism defines activity of human serine protease HtrA3

“…In the bacterial HtrAs such as DegS, DegP and DegQ, it is well documented that their active site pockets get allosterically stabilized or activated by substrate binding at the distal regulatory PDZ domain [59]. For the human HtrAs, an allosteric mode of activation is well established in the literature for all human HtrAs (-1,-2 and -4) except HtrA3 [12,13]. We recently demonstrated that an allosteric mode of regulation exists for HtrA3 through a novel non-canonical pocket spanning residues both in the SPD and PDZ domains [18].…”

“…Human HtrAs are primarily trimeric [13,14] while their prokaryotic counterparts are capable of forming higher-order oligomers [3]. HtrA3 crystal structure represents a trimeric ensemble of three molecules of the monomeric protein where three phenylalanine residues (F140, F142 and F255) from each monomeric chain 'lock' together to stabilize the trimer.…”

“…This might point towards additional roles of HtrA3 in protein quality control under cellular stress conditions such as heat shock which is a primary function in its homologs, especially HtrA2 [66]. HtrA3, with a melting temperature of 63°C has more stability compared with HtrA4 (58°C) but less than that of HtrA2 (73°C) [13,14] suggesting that the conformational changes induced in these proteins by temperature might be different.…”

“…Among human HtrAs, HtrA2 in particular, shows a significant increase in activity with temperature [13,14]. This heat activation is associated with considerable plasticity at the PDZ-protease interface similar to activation via substrate binding at PDZ [12,14].…”

“…While HtrA1 and HtrA2 have been extensively characterized, HtrA3 and -4 are the relatively lesser studied members of this family. Briefly, literature available till date reports that the HtrA homologs assume a trimeric pyramidal architecture and exhibit similar mechanism of activation [11][12][13][14][15][16][17]. While HtrA-1, -2 and -4 have been reported to be allosterically activated [12,14,16], only one such study reports allostery in HtrA3 till date [18].…”