Biology subjects

Paknejad, N.

Publications and source records attributed to Paknejad, N..

6 recordsLinked to original sources

bioRxiv2026

Lipid Activation of a Thalamic GPCR Extends Working Memory Time-scales

Working memory capacity is poorly understood. The ability to control and extend working memory time-scales provides the potential to alleviate cognitive decline in disease and aging. Through an unbiased genetic search, we previously identified a thalamic orphan receptor Gpr12 as a potent enhancer of working memory, however its activation mechanism remains poorly understood. Here, we describe the CryoEM structure of Gpr12, revealing a lipidic regulatory site enabling an activated signaling state. By surveying the native lipidic environment in mouse thalamus we identified a class of 20 carbon:4 double bond fatty acid eicosanoids as potential ligands. Cell-based assays confirmed that the endogenous cannabinoid anandamide (AEA), but not other closely related family members or derivatives, robustly activates Gpr12. In vivo imaging during behavior revealed that Gpr12 activation produces a striking molecular state - the persistent suppression of cAMP in thalamus that tracks the duration of memory maintenance. Notably, genetic or pharmacological manipulations that enhance the AEA-Gpr12 signaling axis are sufficient to prolong cAMP suppression and extend the temporal window of memory maintenance. Furthermore, AEA-mediated cAMP suppressions in thalamus support sustained neural activity in PFC, specifically during memory maintenance. Thus, while cannabinoids often impair memory, here we identify an AEA-Gpr12 signaling axis in thalamus that enhances memory, including in primates. These findings identify a lipidic signaling mechanism in thalamus that is sufficient to control and extend working memory duration.

neuroscience↗

bioRxiv2026

The structural logic of insect olfactory receptor assembly and gating

Insects detect the chemical world using a large family of odorant-gated ion channels, each formed from a variable odorant-binding subunit (OR) and a single conserved co-receptor Orco. This modular organization is thought to allow tuning ORs to diversify their chemical recognition while Orco provides structural stability to the heteromer. Yet Orco can be autonomously activated by synthetic agonists, suggesting that it may contribute to channel gating rather than serving solely as a structural scaffold. Here, we combine cryo-electron microscopy with analyses of receptor stoichiometry and function to define the structural logic underlying Orco-OR assembly and gating. We show that Orco retains the canonical ligand-binding pocket of ORs but is chemically insulated from environmental odorants by a phospholipid that occupies this site. The Orco agonist VUAA4 instead binds a membrane-accessible crevice adjacent to the gate, defining a distinct site of allosteric modulation. We further demonstrate that Orco-OR heteromers can assemble in multiple stoichiometries through shape complementarity within the intracellular anchor domain and resolve structures with both a 3:1 and 2:2 architecture. Receptors constrained to a 2:2 stoichiometry are functional but productive gating requires cooperative engagement of multiple subunits within the heteromer. Comparison with the distinct gating states of a basal homomeric olfactory receptor suggests that existing Orco-OR structures capture nonconductive intermediates within the broader conformational landscape of this receptor family. Together, these findings suggest how Orco can flexibly assemble and function with highly divergent ORs, acting not simply as a structural scaffold but as an integral partner in cooperative channel gating, thereby enabling the extraordinary diversification of insect olfactory receptors.

neuroscience↗

bioRxiv2026

Structural dynamics of adenine nucleotide potentiation of the human type 2 IP3 receptor

Inositol trisphosphate receptors (IP3R) are intracellular calcium (Ca2+) channels that mediate Ca2+ flux from the endoplasmic reticulum (ER) into the cytosol, playing a critical role in Ca2+ signaling. IP3R activity requires IP3 and Ca2+ and is potentiated by adenine nucleotides through a poorly understood mechanism. Here, we combined single-particle cryo-electron microscopy and all-atom molecular dynamics simulations to investigate the potentiation of IP3Rs by adenine nucleotides. Our structures reveal that ATP and cAMP bind to a conserved site in the juxtamembrane domain, which connects the cytoplasmic IP3- and Ca2+-binding sites with the transmembrane pore. Molecular dynamics simulations predict that the binding of adenine nucleotides rigidifies the juxtamembrane domain, primarily through the coordination of the adenine base. Consistent with the adenine base being critical for potentiation, mutations that disrupt the interactions with the adenine base perturb Ca2+ flux in cells. Taken together, our data suggest that adenine nucleotides potentiate IP3R channel activity by rigidifying the juxtamembrane domain to improve coupling between IP3 and Ca2+ binding and pore opening. Significance StatementInositol-1-4-5-trisphosphate receptors (IP3Rs) are the main intracellular calcium (Ca2+) release channels in non-excitable cells and contribute significantly to intracellular Ca2+ release in excitable cells. Regulation of IP3Rs by inositol-1-4-5-trisphosphate (IP3), adenine nucleotides, and Ca2+ is fundamental to both intracellular Ca2+ homeostasis and signaling. We show that adenine nucleotides modulate IP3R activity by tuning the dynamics of a mechanical fulcrum-like domain, the juxtamembrane domain (JD), which physically couples the IP3- and Ca2+-binding sites in the large regulatory cytoplasmic domain to the channel pore. Using a combination of structural, computational, and functional studies, we show that adenine nucleotides bind to and restrict the movement of the JD of the human type 2 IP3R (hIP3R2). We find that that the coordination of adenine nucleotides is primarily driven by hydrophobic interactions with the adenine moieties of the nucleotides and that these interactions are critical for normal hIP3R2 function. This work establishes the foundation for further research into the physiological role of adenine nucleotide modulation of IP3Rs.

biophysics↗

bioRxiv2022

Structural titration reveals Ca2+-dependent conformational landscape of the IP3 receptor

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+-permeable cation channels whose biphasic dependence on cytoplasmic Ca2+ gives rise to cytosolic Ca2+ oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP3R-mediated Ca2+ oscillations, the structural underpinnings of the biphasic Ca2+ dependence that underlies Ca2+ oscillations are incompletely understood. Here, we collected images of an IP3R with Ca2+ at concentrations spanning five orders of magnitude. Unbiased image analysis revealed that Ca2+ binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for free energy, we demonstrate that Ca2+ binding at a high-affinity site allows IP3Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca2+, IP3Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca2+ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca2+-dependence of IP3R channel activity.

biophysics↗

bioRxiv2022

Allosteric interactions prime androgen receptor dimerization and activation

The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how ARs individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging ARs domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by ARs oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of ARs cooperative interactions translate to consequences in development and disease.

biochemistry↗

bioRxiv2019

Cell-projection pumping: A hydrodynamic cell-stiffness dependent mechanism for cytoplasmic transfer between mammalian cells

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fib) and malignant cells (MC). Others report similar transfer via either tunneling nanotubes (TNT) or shed membrane vesicles and this changes the phenotype of recipient cells. Our current time-lapse microscopy showed most exchange was from Fib into MC, with less in the reverse direction. Although TNT were seen, we were surprised transfer was not via TNT, but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless, by their organellar cargo and the grooves they formed indenting MC, while this was consistent with holotomography. Discrete, rapid and highly localized transfer events, evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell-projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell-projections normally returns cytoplasm to the cell body. We hypothesize cell-projection pumping (CPP), where cytoplasm in retracting cell-projections partially equilibrates into adjacent recipient cells via micro-fusions that form temporary inter-cellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modelling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure towards least resistance. Predictions from the model were satisfied when Fib were co-cultured with MC, and fluorescence exchange related with cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MC or Fib was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian inter-cellular cytoplasmic transfer and communication. SIGNIFICANCETime-lapse observations of co-cultured cells led us to hypothesize what we believe to be a novel hydrodynamic mechanism transferring cytoplasm between cells. Similar transfer by other mechanisms markedly affects cell behavior. Combined mathematical modelling, satisfaction of predictions from the mathematical model in cell culture experiments, and separate computer simulations that generate outcomes similar to experimental observations, support our hypothesized mechanism.

cell biology↗