Biology subjects

Itskanov, S.

Publications and source records attributed to Itskanov, S..

7 recordsLinked to original sources

bioRxiv2026

Dynamin-2 promotes Atg9A retrieval from phagophores during autophagy.

Autophagy involves the rapid growth of phagophores through membrane addition. This growth is triggered by vesicles containing the Atg9A protein. However, Atg9A is not incorporated into mature autophagosomes. We now demonstrate that Dynamin-2 (Dnm2) colocalizes with the BAR domain protein Endophilin-B1 (EndoB1/Bif-1/SH3GLB1) and other autophagy proteins when autophagy is induced. Our data suggest that Atg9A is retrieved from phagophores via fission, with Dnm2 acting as the membrane scission protein. Blocking Atg9A recycling, either by mutating Dnm2, using RNA interference, or applying chemical inhibitors, results in Atg9A remaining in autophagosomes and being degraded during autophagy. Overall, these findings provide new insights into the roles of membrane-scission proteins in autophagy.

cell biology↗

bioRxiv2025

A structural roadmap for the formation of the coronavirus nsp3/nsp4 double membrane vesicle pore and its implications for polyprotein processing and replication/transcription

Coronavirus replication is understood to occur within double membrane vesicles (DMVs) that arise during viral infection. Prior work has determined that these DMVs have characteristic pores formed from the non-structural viral proteins nsp3 and nsp4, which facilitate export of newly synthesized viral RNA. Yet how the replication machinery, which is comprised of the non-structural proteins nsp7 to nsp16, is recruited to the DMV remains a mystery. Working from AlphaFold and previously determined structures, we constructed a series of models that link formation of the DMV pore to nsp5 protease processing of the polyprotein and trapping of the cleaved products within the DMV itself. We argue that the initial pore is formed from twelve subunits of nsp3 and six subunits each of the intermediate uncleaved polyproteins pp1a (nsp4-nsp10) and pp1ab (nsp4-nsp16). Formation of this initial structure activates the protease function of alternating nsp5 subunits within a close-packed ring, facilitating the initial trans- cleavage of the nsp4-nsp5 linkage. Maturation of the pore follows, as does formation of canonical nsp5 dimers, which can process the remainder of the polyproteins. When protease activation occurs subsequent to closure of the DMV, the cleavage products, whose stoichiometry is consistent with the previously proposed nsp15-centered hexameric replication complex, will be trapped inside. ImportanceCoronaviruses, like many other positive-sense single-stranded RNA viruses, rely on intracellular membrane remodeling to create a protected environment for efficient replication to occur. The resulting double membrane vesicles (DMVs) have characteristic pores formed from the non-structural viral proteins nsp3 and nsp4, while enzymes responsible for RNA replication, including the polymerase nsp12, are contained inside. Recent structural work has elucidated the nature of the pores, but how the polymerase and other viral proteins are recruited to the DMV represents a major gap in our current knowledge. Here we present a novel, step-by-step structural model of how the pores initially form from the uncleaved polyprotein, how protease activity is initiated, and how the viral replication machinery is trapped within the DMV.

molecular biology↗

bioRxiv2024

Mfn2 induces NCLX-mediated calcium release from mitochondria.

Mfn2 is a mitochondrial outer-membrane fusion protein that also functions as a tether between mitochondria and the ER. Here, we identify a previously unrecognized role for Mfn2 in promoting mitochondrial Ca2+ release via the Na+/Ca2+ exchanger NCLX. This function was uncovered through studies with the fungal toxin phomoxanthone A (PXA), which induces NCLX-dependent Ca2+ release by directly targeting Mfn2. Mfn2-dependent Ca2+ release through NCLX is similarly triggered by ROS in respiring cells treated with oligomycin or mitoPQ. ROS enhances Ca2+ release by strengthening the interaction between Mfn2 and NCLX, an interaction that also requires the mitochondrial outer-membrane protein SLC25A46. Together, these proteins coordinate mitochondrial fission and Ca2+ release to initiate mitophagy. The antioxidant N-acetylcysteine blocks ROS-induced mitochondrial fission, but inhibition of Ca2+ release with the NCLX inhibitor CGP37157 does not, indicating that ROS-driven fission is independent of Ca2+ release. In contrast, Ca2+ release is required for efficient mitophagy, as NCLX inhibition arrests this process at a later stage. We further show that Ca2+ promotes mitophagy through NEDD4-1, which is a Ca2+-responsive E3 ubiquitin ligase. Together, these findings connect mitochondrial ROS production to Ca2+ signaling, mitochondrial remodeling, and mitophagy, providing new insight into how mitochondrial dysfunction may contribute to neurodegenerative and metabolic disease.

cell biology↗

bioRxiv2024

Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10

Doa10 (MARCH6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we defined the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its well-defined degron Deg1. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrates degron peptide into the lateral tunnel is required for efficient polyubiquitination. The N- and C-terminal membrane domains of Doa10 seem to form fence-like features to restrict polyubiquitination to those proteins that can access the central cavity and lateral tunnel.

biochemistry↗

bioRxiv2022

A common mechanism of Sec61 translocon inhibition by small molecules

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of the human Sec61 channel inhibited by a comprehensive panel of structurally distinct small molecules-- cotransin, decatransin, apratoxin F, ipomoeassin F, mycolactone, cyclotriazadisulfonamide (CADA) and eeyarestatin I (ESI). Remarkably, all inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of the channel. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain of Sec61 in a closed state, thereby preventing the protein-translocation pore from opening. Our study reveals molecular interactions between Sec61 and its inhibitors in atomic detail and offers the structural framework for further pharmacological studies and drug design.

biochemistry↗

bioRxiv2020

Stepwise gating of the Sec61 protein-conducting channel by Sec63 and Sec62

The universally conserved Sec61/SecY channel mediates transport of many newly synthesized polypeptides across membranes, an essential step in protein secretion and membrane protein integration1-5. The channel has two gating mechanisms--a lipid-facing lateral gate, through which hydrophobic signal sequences or transmembrane helices (TMs) are released into the membrane, and a vertical gate, called the plug, which regulates the water-filled pore required for translocation of hydrophilic polypeptide segments6. Currently, how these gates are controlled and how they regulate the translocation process remain poorly understood. Here, by analyzing cryo-electron microscopy (cryo-EM) structures of several variants of the eukaryotic post-translational translocation complex Sec61-Sec62-Sec63, we reveal discrete gating steps of Sec61 and the mechanism by which Sec62 and Sec63 induce these gating events. We show that Sec62 forms a V-shaped structure in front of the lateral gate to fully open both gates of Sec61. Without Sec62, the lateral gate opening narrows, and the vertical pore becomes closed by the plug, rendering the channel inactive. We further show that the lateral gate is opened first by interactions between Sec61 and Sec63 in both cytosolic and luminal domains, a simultaneous disruption of which fully closes the channel. Our study defines the function of Sec62 and illuminates how Sec63 and Sec62 work together in a hierarchical manner to activate the Sec61 channel for post-translational translocation.

biochemistry↗

bioRxiv2018

Dynamin-2 facilitates Atg9 recycling from nascent autophagosomes

Autophagy involves rapid growth of phagophores through membrane addition. Newly added membranes are derived from other organelles through vesicles carrying the Atg9 protein. Membrane is delivered by fusing these vesicles with the phagophores. Atg9 is, nevertheless, not incorporated in autophagosomes. We now show that this protein is retrieved from phagophores by fission utilizing Dynamin-2 (Dnm2) as the membrane scission protein. Blocking Atg9 recycling by interfering with Dnm2 helps retain Atg9 in autophagosomes and degrades this protein by autophagy. Dnm2 colocalizes with the BAR domain protein Endophilin-B1 (EndoB1/Bif-1) when autophagy is induced, consistent with transient interactions during Atg9 retrieval. EndoB1 and Dnm2 also control the downstream fusion of phagophores to late endosomes, thus ensuring the completion of phagophores before proceeding to the next stage in the autophagy process. These data provide novel insights into the roles of membrane scission proteins during autophagy.

cell biology↗