The effect of chrysin binding on the conformational dynamics and unfolding pathway of human serum albumin.

Studies on the interactions between ligands and proteins provide insights into how a possible medication alters the structures and activities of the target or carrier proteins. The natural flavonoid aglycone Chrysin (CHR) has demonstrated anti-inflammatory, antioxidant, antiapoptotic, neuroprotective, and antineoplastic effects, both in vitro and in vivo. In this work, we investigated the impact of CHR binding on the as-yet-unexplored conformation, dynamics, and unfolding mechanism of human serum albumin (HSA). We determined CHR binding to HSA domain-II with the association constant (Ka) of 2.70 ± 0.21 × 105 M-1. The urea-induced sequential unfolding mechanism of HSA was used to elucidate the debatable binding location of CHR. CHR binding induced both secondary and tertiary structural alterations in the protein as studied by far-UV circular dichroism and intrinsic fluorescence spectroscopy. Red edge excitation shift (REES) indicated a decrease in conformational dynamics of the protein on the complex formation. This suggested an ordered compact and spatial arrangement of the CHR-boundmolecule. The binding of CHR was found to significantly modulate the urea-induced unfolding pathway of HSA. Urea-induced unfolding pathway of HSA became a two-state process (N-U) from a three-state process (N-I-U). The interaction of CHR is found to increase the thermal stability of the protein by ∼4 °C. This study focuses on the fundamental sciences and demonstrates how prospective medication compounds can alter the dynamics and stability of protein structure.

Plumbagin accelerates serum albumin's amyloid aggregation kinetics and generates fibril polymorphism by inducing non-native ß-sheet structures.

The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant Plumbago zeylanica, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant Ka âˆ¼ 10-4 M-1)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of ß-sheet (∼18%) and disordered (∼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native ß-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.

5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling.

Receptor-interacting protein kinases (RIPK)-1 and -3 play crucial roles in cell fate decisions and are regulated by multiple checkpoint controls. Previous studies have identified IKK1/2- and p38/MK2-dependent checkpoints that phosphorylate RIPK1 at different residues to inhibit its activation. In this study, we investigated TNF-induced death in MAPK-activated protein kinase 2 (MK2)-deficient cells and found that MK2 deficiency or inactivation predominantly leads to necroptotic cell death, even without caspase inhibition. While RIPK1 inhibitors can rescue MK2-deficient cells from necroptosis, inhibiting RIPK3 seems to switch the process to apoptosis. To understand the underlying mechanism of this switch, we screened a library of 149 kinase inhibitors and identified the adenosine analog 5-Iodotubercidin (5-ITu) as the most potent compound that sensitizes MK2-deficient MEFs to TNF-induced cell death. 5-ITu also enhances LPS-induced necroptosis when combined with MK2 inhibition in RAW264.7 macrophages. Further mechanistic studies revealed that 5-ITu induces RIPK1-dependent necroptosis by suppressing IKK signaling in the absence of MK2 activity. These findings highlight the role for the multitarget kinase inhibitor 5-ITu in TNF-, LPS- and chemotherapeutics-induced necroptosis and its potential implications in RIPK1-targeted therapies.

PRMT5-mediated regulatory arginine methylation of RIPK3.

The TNF receptor-interacting protein kinases (RIPK)-1 and 3 are regulators of extrinsic cell death response pathways, where RIPK1 makes the cell survival or death decisions by associating with distinct complexes mediating survival signaling, caspase activation or RIPK3-dependent necroptotic cell death in a context-dependent manner. Using a mass spectrometry-based screen to find new components of the ripoptosome/necrosome, we discovered the protein-arginine methyltransferase (PRMT)-5 as a direct interaction partner of RIPK1. Interestingly, RIPK3 but not RIPK1 was then found to be a target of PRMT5-mediated symmetric arginine dimethylation. A conserved arginine residue in RIPK3 (R486 in human, R415 in mouse) was identified as the evolutionarily conserved target for PRMT5-mediated symmetric dimethylation and the mutations R486A and R486K in human RIPK3 almost completely abrogated its methylation. Rescue experiments using these non-methylatable mutants of RIPK3 demonstrated PRMT5-mediated RIPK3 methylation to act as an efficient mechanism of RIPK3-mediated feedback control on RIPK1 activity and function. Therefore, this study reveals PRMT5-mediated RIPK3 methylation as a novel modulator of RIPK1-dependent signaling.