Comparative analysis of canine and human HtrA2 to delineate its role in apoptosis and cancer.

Therapeutically, targeting the pro- and anti-apoptotic proteins has been one of the major approaches behind devising strategies to combat associated diseases. Human high-temperature requirement serine protease A2 (hHtrA2), which induces apoptosis through both caspase-dependent and independent pathways is implicated in several diseases including cancer, ischemic heart diseases, and neurodegeneration, thus making it a promising target molecule. In the recent past, the canine model has gained prominence in the understanding of human pathophysiology that was otherwise limited to the rodent system.

Targeting type I DED interactions at the DED filament serves as a sensitive switch for cell fate decisions.

Activation of procaspase-8 in the death effector domain (DED) filaments of the death-inducing signaling complex (DISC) is a key step in apoptosis. In this study, a rationally designed cell-penetrating peptide, DEDid, was engineered to mimic the h2b helical region of procaspase-8-DED2 containing a highly conservative FL motif. Furthermore, mutations were introduced into the DEDid binding site of the procaspase-8 type I interface.

cFLIP - An interacting partner and a novel substrate for pro-apoptotic serine protease HtrA2.

Background: HtrA2, a pro-apoptotic protease, plays a crucial role in apoptosis by cleaving inhibitory and anti-apoptotic proteins by translocating from mitochondria to the cytosol. Prior studies in ischemic cells have indicated that cytosolic HtrA2 triggers cFLIP degradation, plausibly through direct interaction. In this study, we have characterized the cFLIP protein, validated its interaction with HtrA2, and demonstrated that cFLIP is also a substrate of HtrA2.

Elucidating the interaction of C-terminal domain of Vaccinia-Related Kinase 2A (VRK2A) with B-cell lymphoma-extra Large (Bcl-xL) to decipher its anti-apoptotic role in cancer.

Vaccinia-Related Kinase 2 (VRK2) is an anti-apoptotic Ser/Thr kinase that enhances drug sensitivity in cancer cells. This protein exists in two isoforms: VRK2A, the longer variant, and VRK2B, which lacks the C-terminal region and transmembrane domain. While the therapeutic importance of VRK2 family proteins is known, the specific roles of VRK2A and its interplay with apoptotic regulator Bcl-xL (B-cell lymphoma-extra Large) remain elusive.

Purification of hetero-oligomeric protein variants using a modified tandem affinity purification approach.

Here, we describe a protocol for artificially generating hetero-oligomeric protein complexes from the homo-oligomers using a sequential denaturation-renaturation strategy, followed by a modified affinity chromatography protocol used for their purification. This protocol enables one to obtain a homogenous population of hetero-oligomers and understand the contribution of each protomer through further biochemical and/or biophysical characterization. For complete details on the use and execution of this protocol, please refer to Parui et al.

A comparative in silico study to detect the effect of food-additives on metabolic protein and its perturbations compensated by osmolytes.

Since its inception, food additive has been an integral part of the food processing industry with various commercial roles. Besides its advantages, various studies have already highlighted its long-term adverse effects on human health. However, in terms of protein structures and functions, the innate mechanism that triggers these effects has not been elucidated in previously reported studies.

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

Vaccinia-related kinases (VRK) are serine-threonine kinases that regulate several signaling pathways. The isoform-VRK2A of one such kinase VRK2 controls cell stress response by interacting with TAK1, a mitogen-activated protein 3 kinase (MAP3K), via its partly cytosolic C-terminal transmembrane domain (VTMD). To establish the driving force and identify the key residues of the VRK2A-TAK1 interaction, we expressed and purified the standalone 3.

Unraveling the Dichotomy of Enigmatic Serine Protease HtrA2.

Mitochondrial high-temperature requirement protease A2 (HtrA2) is an integral member of the HtrA family of serine proteases that are evolutionarily conserved from prokaryotes to humans. Involvement in manifold intricate cellular networks and diverse pathophysiological functions make HtrA2 the most enigmatic moonlighting protease amongst the human HtrAs. Despite perpetuating the oligomeric architecture and overall structural fold of its homologs that comprises serine protease and regulatory PDZ domains, subtle conformational alterations and dynamic enzymatic regulation through the distinct allosteric mode of action lead to its functional diversity.

Role of conserved regulatory loop residues in allosteric propagation of serine protease HtrA2.

High temperature requirement protease A2 (HtrA2) is a mitochondrial serine protease that demonstrates multifaceted roles including protein quality control and proapoptotic properties in humans, making it a potential therapeutic target. Current literature suggests involvement of flexible regulatory loops in governing the allosteric propagation within the trimeric HtrA2 ensemble. Here, we have identified three important residues - R147, P148 (L3 loop) and F131 (LD loop) surrounding the catalytic-site that play crucial roles in stabilizing HtrA2 active conformation during its multimodal activation.

Regulation of extrinsic apoptotic signaling by c-FLIP: towards targeting cancer networks.

The extrinsic pathway is mediated by death receptors (DRs), including CD95 (APO-1/Fas) or TRAILR-1/2. Defects in apoptosis regulation lead to cancer and other malignancies. The master regulator of the DR networks is the cellular FLICE inhibitory protein (c-FLIP).

Remodelling structure-based drug design using machine learning.

To keep up with the pace of rapid discoveries in biomedicine, a plethora of research endeavors had been directed toward Rational Drug Development that slowly gave way to Structure-Based Drug Design (SBDD). In the past few decades, SBDD played a stupendous role in identification of novel drug-like molecules that are capable of altering the structures and/or functions of the target macromolecules involved in different disease pathways and networks. Unfortunately, post-delivery drug failures due to adverse drug interactions have constrained the use of SBDD in biomedical applications.

Loss of GSK-3β mediated phosphorylation in HtrA2 contributes to uncontrolled cell death with Parkinsonian phenotype.

HtrA2, a proapoptotic mitochondrial serine protease, promotes cellular protection against oxidative damage. Literature reports show positive correlation between loss of HtrA2 protease activity and Parkinson's Disease (PD) susceptibility. Homozygous loss-of-function mutations in murine-HtrA2, and when they rarely occur in humans result in severe neurodegeneration and infantile death.

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

HtrA2 (high-temperature requirement A2) and GRIM-19 (gene associated with retinoic and interferon-induced mortality 19 protein) are involved in various biological functions with their deregulation leading to multiple diseases. Although it is known that the interaction between GRIM-19 with HtrA2 promotes the pro-apoptotic activity of the latter, the mechanistic details remained elusive till date. Moreover, designing allosteric modulators of HtrA2 remains obscure due to lack of adequate information on the mode of interaction with its natural substrates cum binding partners.

Dual specificity phosphatase 9: A novel binding partner cum substrate of proapoptotic serine protease HtrA2.

Human high temperature requirement protease A2 (HtrA2) is a trimeric PDZ bearing proapoptotic serine protease, which is involved in various cellular processes and pathologies. Research in the last decade strongly advocates its role as a potential therapeutic target and therefore warrants the need to minutely investigate its mechanism of action, regulation, interactions with other proteins and its binding specificities. In this particular study, we adopted an in silico approach to predict novel interacting partners and/or substrates of HtrA2 by building a peptide library using a binding pattern search.

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

Human HtrA3 (high-temperature requirement protease A3) is a trimeric multitasking propapoptotic serine protease associated with critical cellular functions and pathogenicity. Implicated in diseases including cancer and pre-eclampsia, its role as a tumor suppressor and potential therapeutic target cannot be ignored. Therefore, elucidating its mode of activation and regulatory switch becomes indispensable towards modulating its functions with desired effects for disease intervention.

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

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.

Identification of a distal allosteric ligand binding pocket in HtrA3.

Human HtrA3 (High temperature requirement protease A3) is a trimeric PDZ bearing propapoptotic serine protease, which is involved in various diseases including cancer and pre-eclampsia. Proposed to be a tumor suppressor, its role as a potential therapeutic target is strongly advocated. Therefore, it becomes imperative to gain insights into its mechanism of action and regulation.

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

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.

Correction to: Histone isoform H2A1H promotes attainment of distinct physiological states by altering chromatin dynamics.

After publication of this article [1], it was noticed Duane Smoot and Hassan Ashktorab who made and provided the cell line HFE145 were not included in the author list.

An efficient method for FITC labelling of proteins using tandem affinity purification.

Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches.