Biology subjects

Kutti, V. R.

Publications and source records attributed to Kutti, V. R..

3 recordsLinked to original sources

bioRxiv2022

Structural insights into pore dynamics of human Pannexin isoforms

Pannexins are single-membrane large-pore ion channels that release ATP upon activation. Three isoforms of pannexins, 1, 2, and 3, perform diverse cellular roles, including inflammation, differentiation, neuropathic pain, and ATP release. In this study, we report the cryoEM structure of pannexin 3 at 3.9 [A] and characterize the structural differences with pannexin isoforms 1 and 2. We observe the organization of the Pannexin 3 vestibule into two distinct chambers with a wider pore radius in comparison to both PANX1 and 2 isoforms. We further report the structure of pannexin1 congenital mutant R217H in the resolution range of 3.9 [A]. The congenital mutant R217H in transmembrane helix3 (TM3), R217H induce structural changes that leads to a partially closed pore and altered ATP interaction propensities. The channel conductance of the congenital mutant displays weakened voltage sensitivity. The results showcase a complete comparison of the three pannexin isoform structures that along with the structure of Pannexin 1 congenital mutant highlight distinct structural features of pannexin isoforms and the allosteric role of distant substitutions in dictating channel behavior in Pannexin 1.

biochemistry↗

bioRxiv2021

Substrate dependent homeostatic control of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE) from Mycobacterium smegmatis

Cyclic-di-nucleotide based secondary messengers regulate various physiological processes including the stress responses in bacteria. In the past decade, cyclic diadenosine monophosphate (c-di-AMP) has emerged as a crucial second messenger, implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, sporulation etc. The level of c-di-AMP is maintained in the cell by the action of two opposing enzymes, namely diadenylate cyclase (DAC) and phosphodiesterase (PDE). In mycobacteria, this molecule is essential for its regulatory role in bacterial physiology and host-pathogen interactions. However, such modulation of c-di-AMP remains to be explored in Mycobacterium smegmatis. Here, we systematically characterised the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pH and osmolytic conditions in vitro. Our biochemical assays show that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity in vivo increases significantly, strengthening this observation. However, under physiological conditions, the activity of MsDisA was moderate with the formation of intermediates. To get further insights into the structural characteristics, we determined the cryo-EM structure of the MsDisA, revealing some interesting features. Biochemical analysis of individual domains shows that the N-terminal minimal region alone can form a functional octamer. Altogether, our results reveal the biochemical and structural regulation of mycobacterial c-di-AMP in response to various environmental stress.

biophysics↗

bioRxiv2021

Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H2O2 generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (Claspin/ATRIP/TOPBP1), can induce phosphorylation of Chk1 in response to micromolar concentrations of H2O2 in minutes. The findings presented reveal that H2O2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.

developmental biology↗