Biology subjects

Torosyan, H.

Publications and source records attributed to Torosyan, H..

4 recordsLinked to original sources

bioRxiv2025

Structures of the PI3Kα/KRas complex on lipid bilayers reveal the molecular mechanism of PI3Kα activation

PI3K is a potent oncogene that converts PIP2 to PIP3 at the plasma membrane upon activation by receptor tyrosine kinases and Ras GTPases. In the absence of any structures of activated PI3K, the molecular details of its activation remain unknown. Here, we present cryo-EM structures of the PI3K/KRas complex embedded in lipid nanodiscs, revealing a rich ensemble of PI3K states adopted at the membrane surface. The sequential addition of a lipid bilayer, PIP2 and an activating phosphopeptide leads to the progressive release of key inhibitory domains from the PI3K catalytic core, which directly correlates with the reorganization of its active site. While association with POPC/POPS nanodiscs partially relieves PI3K autoinhibition, incorporation of PIP2 triggers near-complete displacement of PI3K inhibitory domains and significant restructuring of active site regulatory motifs. The addition of the activating phosphopeptide induces dimerization of the PI3K/KRas complex through a p110 catalytic subunit-mediated interface that is sterically occluded in autoinhibited PI3K. In cells, this dimeric PI3K complex amplifies Akt signaling in response to growth factor stimulation. Collectively, our structures map the conformational landscape of PI3K activation and reveal previously unexplored interfaces for potential therapeutic targeting.

biophysics↗

bioRxiv2022

Structural insights into regulation of the PEAK3 scaffold by 14-3-3

The three members of the PEAK family of pseudokinases (PEAK1, PEAK2, and PEAK3) are molecular scaffolds that have recently emerged as important regulatory nodes in signaling pathways that control cell migration, morphology, and proliferation, and they are increasingly found to be mis-regulated in human cancers. While no structures of PEAK3 have been solved to date, crystal structures of the PEAK1 and PEAK2 pseudokinase domains revealed their dimeric organization. It remains unclear how dimerization plays a role in PEAK scaffolding functions, as no structures of PEAK family members in complex with their binding partners have been solved. Here, we report the cryo-EM structure of the PEAK3 pseudokinase, also adopting a dimeric state, and in complex with an endogenous 14-3-3 heterodimer purified from mammalian cells. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the Split HElical Dimerization (SHED) domain of the PEAK3 dimer. The binding interface is comprised of a canonical primary interaction involving two phosphorylated 14-3-3 consensus binding sites located in the N-terminal domains of the PEAK3 monomers docked in the conserved amphipathic grooves of the 14-3-3 dimer, and a unique secondary interaction between 14-3-3 and PEAK3 that has not been observed in any previous structures of 14-3-3/client complexes. Disruption of these interactions results in the relocation of PEAK3 to the nucleus and changes its cellular interactome. Lastly, we identify Protein Kinase D as the regulator of the PEAK3/14-3-3 interaction, providing a mechanism by which the diverse functions of the PEAK3 scaffold might be fine-tuned in cells.

biophysics↗

bioRxiv2021

CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids

Introductory paragraphThe Hedgehog (HH) pathway is critical for development and adult tissue homeostasis1. Aberrant HH signaling can cause congenital malformations, such as digit anomalies and holoprosencephaly2, and other diseases, including cancer3. Signal transduction is initiated by HH ligand binding to the Patched 1 (PTCH1) receptor on primary cilia, thereby releasing inhibition of Smoothened (SMO), a HH pathway activator4. Although cholesterol and several oxysterol lipids, which are enriched in the ciliary membrane, play a crucial role in HH activation4,5, the molecular mechanisms governing the regulation of these lipid molecules remain unresolved. Here, we identify Canopy 4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that controls membrane sterol lipid levels. Cnpy4-/- embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway at the level of SMO in vitro, and elevates membrane levels of accessible sterol lipids such as cholesterol, an endogenous ligand involved in SMO activation6. Thus, our data demonstrate that CNPY4 is a negative regulator that fine-tunes the initial steps of HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.

biochemistry↗

bioRxiv2020

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of -synuclein, a dynamic protein linked to Parkinsons disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of -synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to -synuclein biology and pathobiology. We found that the conformation of -synuclein previously shown to drive yeast toxicity--an extended, membrane-bound helix--is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress -synuclein toxicity. Moreover, we find that sequence determinants of -synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that -synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

biochemistry↗