Biology subjects

Nayak, T. K.

Publications and source records attributed to Nayak, T. K..

6 recordsLinked to original sources

bioRxiv2026

Allosteric signal initiation and communication in neuromuscular acetylcholine receptors

Acetylcholine receptors (AChRs) expressed at the nerve-muscle synapses are prototypic allosteric receptors that shuttle between a resting Closed (C) and active Open (O) states. ACh binding at the neurotransmitter binding sites (TBS) leads to opening of the gate in the channel pore that regulates ion flow. How agonist binding to the TBS communicates the allosteric signal to the channel gate located [~]50 [A] away is not understood. In the absence of agonists, the wild-type and mutant AChRs, including those causing congenital myasthenia syndrome, show constitutive gating. However, whether allosteric activation pathways in the presence vs absence of agonists are identical, is debatable. Here, by using a combination of kinetic modelling, phi ({phi})- and activation energy ({Delta}{Delta}G[palclick]) estimation for >60 residues from single channel current recordings and molecular dynamics simulations, we show the existence of parallel gating pathways ( major and minor) in the unliganded AChRs and activation pathways are non-identical for the liganded vs unliganded AChRs. Kinetic analysis of the minor gating and correlation between the state residence probabilities vs C-loop conformations suggest, 1. the C-loop capping triggers allosteric communication independent of the presence of an agonist and, 2. the minor gating is a pre-existing allosteric pathway which is preferentially chosen in the presence of agonists. Further, we show the presence of a continuous live-wire like allosteric network in the liganded receptor between the TBS and the gate constituted by residues which lower the activation energy barrier by >-4 kcal/mol. In contrast, in the unliganded AChRs, the major gating seems to initiate from hub residues in the allosteric network. The results presented here provide novel insight into the mechanism of allosteric signal initiation and communication in AChRs.

biophysics↗

bioRxiv2025

TRPML3 regulates neuronal gene expression in an in vitro model of autophagy and may act as a genetic marker of familial neurodegenerative disorders

Autophagy is a conserved pro-survival pathway for delivering misfolded proteins and damaged organelles to lysosomes for degradation and protein homeostasis. Anomaly in autophagy leads to aberrant protein aggregation in neuronal cells, which is a common etiology of neurodegenerative disorders. Endo-lysosomal cation channel TRPML3 (Transient Receptor Potential Mucolipin-3) has been shown to induce autophagy in cell line models. However, the mechanism of TRPML3 mediated autophagy induction and the underlying gene expression changes are not clearly understood. Here, by using Ca2+-and electrical-current measurements, RNA sequencing and RT PCR studies, we explored the cellular function of TRPML3 and the global transcriptomic profile in a cell-based serum starvation model of autophagy. We report that serum starvation leads to downregulation of neuronal developmental genes during autophagy induction. TRPML3 overexpression further amplifies the effect of starvation in downregulating neuronal gene expression. But, when nutrition is not a limiting condition, TRPML3 overexpression upregulated neuronal genes including those responsible for axon guidance, synaptogenesis, and dendritic arborization. TRPML3 mediated neuronal gene expression changes were, presumably, due to transcription factors (TF) TFEB, FOXO1 and neuron-specific TFs such as SOX2, and ETV5. To further validate the role of TRPML3 in neuronal gene regulation, we performed meta-analysis of publicly available RNAseq datasets on neurodegenerative disorders which provided insight into the heterogeneity in the molecular mechanisms of autophagy and corroborated the TFEB-mediated autophagy induction and neuronal gene expression in TRPML3 overexpression condition. Based on our results, we propose that TRPML3 may act as a potential genetic marker for familial neurodegenerative disorders.

neuroscience↗

bioRxiv2024

Role of surface negative charges in agonist binding to the unliganded open state of the neuromuscular acetylcholine receptor

Acetylcholine receptors (AChRs) expressed at the nerve muscle junction (NMJ) synapses are hetero-pentameric ligand-gated ion channels with two neurotransmitter binding sites (TBS) at -{delta} and -{varepsilon} (adult)/{gamma} (fetal) subunit interfaces. They are typical allosteric proteins which reside in at least two stable conformations: resting (Closed) and active (Open) states. Mechanism of agonist (A) binding to the C state has been extensively studied, but agonist association to the O state has not been clearly understood. Here, by using engineered constitutively active AChRs, single-channel patch-clamp, and molecular dynamics (MD) simulations, we elucidate the differences between unliganded (O) vs liganded (AO) active states and the mechanism of agonist association to the O state. Our results indicate that: 1. At the {gamma}-binding site, for a series of agonists, the O state agonist association rate (jon) was [~]30 times faster than the diffusion limit and agonist affinity was 2-orders of magnitude higher vs at the {delta}- and {varepsilon}-binding sites. 2. Electrostatic surface charge density owing to 4 negatively charged residues ({gamma}E57, {gamma}D113, {gamma}D174, and {gamma}E180) facilitates loop C capping, compaction of the TBS, and agonist stabilization in the AO state. 3. Mutating these residues in combination reversed the jon and binding affinity to those comparable to the {delta}- and {varepsilon}-binding sites. 4. {varphi}-value analysis indicated the presence of a transition state intermediate between the O and AO states. Overall, we elucidate the role of neighboring charged residues outside the TBS in determining high-affinity agonist binding and their significance in shaping the synaptic response at the NMJ. SummaryNicotinic acetylcholine receptors (AChRs) are allosteric proteins that are crucial for muscle contraction. The isomerization of resting closed (C) to active open (O) state can be achieved by both bind-gate and gate-bind (rare) pathway. Little is known about agonist binding to the unliganded functional O state. Here, we elucidate the nature of unliganded O state and the mechanism of agonist binding to this high-affinity O state by single-channel current recordings, protein engineering, in silico methods and phi-analysis. Our results describe an energetic barrier between the unliganded and liganded O state owing to negatively charged amino acids near the agonist site. These charges partially contribute to the low- and high-affinity agonist energies specifically at the embryonic neurotransmitter binding site. We also discussed the physiological significance of these residues in shaping synaptic response. Significance StatementEmbryonic-type AChRs are indispensable for nerve-muscle junction formation. Many mutations in these receptors cause congenital myasthenia syndrome by increasing constitutive channel activation. Therefore, understanding the nature of unliganded functional open (O) state is critical. Here, we describe the nature and the mechanism of agonist binding to the unliganded O (or apo) state. Single-channel currents were recorded with different background constructs and binding site mutations. The results highlight the importance of loop C and ligand orientation in determining high-affinity agonist binding. Further we elucidate the role of electrostatic interactions in agonist binding to the O state (high-affinity binding). We surmise that higher association rate of agonist binding to O state is the reason behind higher efficacy of the fetal receptor.

biophysics↗

bioRxiv2022

DNA damage response signaling is crucial for effective Chikungunya virus replication

Viruses utilize a plethora of strategies to manipulate the host pathways and hijack its machineries for efficient replication. Several DNA as well as handful of RNA viruses are reported to interact with proteins involved in DNA damage responses (DDR). As the DDR pathways have never been explored in Alphaviruses, this investigation intended to determine the importance of the DDR pathways in CHIKV infection through in vitro, in vivo and ex vivo models. The study reveals that CHIKV infection activates the Chk2 and Chk1 proteins associated with DDR signaling pathways and increases DNA damage by 95%. Inhibition of both ATM-ATR kinases by ATM/ATR kinase inhibitor (AAKi) shows drastic reduction in viral particle formation in vitro. Next, the treatment of mice with this drug has been shown to reduce the disease score substantially in CHIKV-infected C57BL/6 mice with 71% decrement in the viral copy and the same has been established in hPBMC-derived monocyte-macrophage populations. Additionally, gene silencing of Chk2 and Chk1 reduces viral progeny formation around 73.7% and 78% respectively. Moreover, it has been demonstrated that CHIKV-nsP2 interacts with Chk2 and Chk1 during CHIKV infection and docking analysis depicts the specific amino acids responsible for these interactions. Further, the data suggests that CHIKV infection induces cell cycle arrest in G1 and G2 phases. In conclusion, this work demonstrated for the first time the mechanistic insight of the induction of DDR pathways by CHIKV that might contribute to the designing of effective therapeutics for the control of this virus infection in future. IMPORTANCEViruses being intra-cellular parasite, need several host cell machineries so as to achieve effective replication of their own genome, along with virus-encoded enzymes. One of the strategies is to hijack the DDR pathways. Several DNA as well as handful of RNA viruses interact with the cellular proteins involved in DDR pathways, however, reports with respect to the association of Chk2 and Chk1 in alphavirus infection are scanty. Hence, this study is amongst the first to report that modulation of DDR pathways is crucial for effective CHIKV infection. This work also shows that there is interaction of CHIKV-nsP2 with two crucial host factors, Chk2 and Chk1 for efficient viral infection. Interestingly, CHIKV infection was found to cause DNA damage and arrest cell cycle in G1 and G2 phases to facilitate viral infection. This information might facilitate to develop effective therapeutics for the control of the CHIKV infection in future.

microbiology↗

bioRxiv2021

MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MK2 and MK3 were selected for further analysis. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation (a) inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during the late phase of CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of this drug against CHIKV. Additionally, CMPD1 also inhibited HSV1 and SARS CoV2-19 infection in vitro. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as key targets for developing effective anti-CHIKV strategies in future. Author summaryChikungunya virus has been a dreaded disease from the first time it occurred in 1952 Tanzania. Since then it has been affecting the different parts of the world at different time periods in large scale. It is typically transmitted to humans by bites of Aedes aegypti and Aedes albopictus mosquitoes. Although, studies have been undertaken to combat the disease still there are no effective strategies like vaccines or antivirals against it. Therefore it is essential to understand the virus and host interaction to overcome this hurdle. In this study two host factors MK2 and MK3 have been taken into consideration to see how they regulate the multiplication of the virus. The in vitro experiments demonstrated that inhibition of MK2 and MK3 restricted viral infection Further, it was observed that this is due to the blocking of lamellipodium formation by modifying the factors involved in the actin cytoskeleton remodeling pathway that is responsible for releasing the virus from the infected cells. Besides, decreased disease score as well as better survivability was noticed in the in vivo experiments with mice. Therefore, MK2 and MK3 could be considered as the key targets for controlling CHIKV infection.

microbiology↗

bioRxiv2019

Autoimmunity Risk Gene IRGM is a Master Negative Regulator of Interferon Response by Controlling the Activation of cGAS STING and RIG-I-MAVS Signaling Pathways

Activation of type 1 interferon response is extensively connected with the antiviral immunity and pathogenesis of autoimmune diseases. Here, we found that IRGM, whose deficiency is linked with the genesis of several autoimmune disorders, is a master negative regulator of the interferon response. Mechanistically, we show that IRGM interacts with nucleic acid sensor proteins, including cGAS and RIG-I, and mediates their autophagic degradation to restrain activation of interferon signaling. Further, IRGM maintains mitophagy flux, and its deficiency results in the accumulation of defunct leaky mitochondria that releases cytosolic DAMPs triggering activation of interferon responses via cGAS-STING and RIG-I-MAVS signaling axis. Due to an enduring type 1 IFN response in IRGM-deficient cells and mice, they were intrinsically resistant to infection of the Japanese Encephalitis virus, Herpes Simplex virus, and Chikungunya virus. Altogether, this study defines the molecular mechanisms by which IRGM maintains interferon homeostasis and protects from autoimmune diseases. Further, it identifies IRGM as a broad therapeutic target for defense against viruses.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC=\"FIGDIR/small/815506v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (48K):\norg.highwire.dtl.DTLVardef@1d4822org.highwire.dtl.DTLVardef@6aebbcorg.highwire.dtl.DTLVardef@19a9b72org.highwire.dtl.DTLVardef@1539195_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗