Biology subjects

Panigrahi, R.

Publications and source records attributed to Panigrahi, R..

4 recordsLinked to original sources

bioRxiv2023

Structural insights into γH2Ax containing nucleosomes

MDC1 is a key mediator of DNA-damage signaling. When DNA double-strand breaks (DSB) occur, the histone variant H2AX on the nucleosome is phosphorylated on its C-terminus at residue Ser139 to form the {gamma}H2AX nucleosome. This phosphorylated form is specifically recognized by the tandem BRCT repeats of MDC1. The MDC1-bound nucleosome serves as a docking platform to promote the localization of other DNA repair factors. To further characterize the nucleosome-BRCT interaction, we developed a time efficient two-step modified native chemical ligation protocol to prepare phosphorylated nucleosomes. Our binding studies show that BRCT interacts with the nucleosome with a higher affinity than the phosphorylated peptide. Using cryogenic electron microscopy (cryo-EM), we obtained structures of the {gamma}H2AX nucleosome revealing the structural basis for nucleosome-nucleosome stacking promoted by interactions of the H4 N-terminal of one nucleosome with its stacked partner. In contrast, we show that binding of the MDC1 BRCT domain disrupts this stacking, suggesting that histone/DNA dynamics are integral to DNA damage signaling.

biochemistry↗

bioRxiv2022

Penetratin inhibits α-synuclein fibrillation and improves locomotor functions in mice model of Parkinsons disease

Parkinsons disease (PD) is the second most common neurodegenerative disease. The presence of lewy bodies, primarily consisting of -synuclein (-syn) aggregates is one of the common features seen in the substantia nigra region of the brain in PD patients. The disease remains incurable and only symptomatic relief is available. We screened various cell-penetrating peptides and reveal that penetratin is a potent inhibitor of -syn aggregation in-vitro, and significantly improved locomotor coordination in mice models of PD in-vivo. The peptide inhibits -syn aggregation in vitro as well as in yeast, and C.elegans models. We further made a cyclic derivative of penetratin by disulfide coupling of N- and C-terminal cysteine residues. Both penetratin and its cyclized derivative interact with -syn. NMR studies show that both linear as well as cyclic derivative interact at the acidic C-terminal tail of the protein. Similar to penetratin, its cyclic derivative inhibited -syn aggregation in the C.elegans model of Parkinsons disease, and also improved worm motility. Molecular Dynamics studies show that penetratin interacts with -synuclein and prevents its conformational transition from disordered into {beta}-sheet rich structure. The therapeutic efficacy of penetratin was further confirmed in a transgenic mice model of the disease, wherein penetratin treatment over a period of 90 days improved locomotor coordination, and halted disease progression. Overall, the present work provides a potent therapeutic agent that could be further explored in the management of PD.

neuroscience↗

bioRxiv2021

Implications of critical nodes-dependent unidirectional cross-talk between Plasmodium and Human SUMO

The endoparasitic pathogen, Plasmodium falciparum (Pf), modulates protein-protein interactions to employ post-translational modifications like SUMOylation in order to establish successful infections. The interaction between E1 and E2 (Ubc9) enzymes governs species specificity in the Plasmodium SUMOylation pathway. Here, we demonstrate that a unidirectional cross-species interaction exists between Pf-SUMO and Human-E2, whereas Hs-SUMO1 failed to interact with Pf-E2. Biochemical and biophysical analysis revealed that surface-accessible Aspartates of Pf-SUMO determine the efficacy and specificity of SUMO-Ubc9 interactions. Furthermore, we demonstrate that critical residues of the Pf-Ubc9 N-terminal are responsible for the lack of interaction between Hs-SUMO1 and Pf-Ubc9. Mutating these residues to corresponding Hs-Ubc9 residues restore electrostatic, {pi}-{pi}, and hydrophobic interactions and allows efficient cross-species interactions. We suggest that the critical changes acquired on the surfaces of Plasmodium SUMO and Ubc9 proteins as nodes can help Plasmodium exploit the host SUMOylation machinery. Thus, Pf-SUMO interactions can be targeted for developing antimalarials.

biochemistry↗

bioRxiv2019

Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

-Synuclein (-Syn) aggregation and amyloid formation is directly linked with Parkinsons disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of -Syn precedes its aggregation. In particular, in vitro generated -Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate -Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote -Syn LLPS and its subsequent maturation. We further demonstrate -Syn liquid droplet formation in cells, under oxidative stress. These cellular -Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured -Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

neuroscience↗