Biology subjects

Singal, B.

Publications and source records attributed to Singal, B..

7 recordsLinked to original sources

bioRxiv2026

The Structure of the Picornaviral 2C:RNA holoenzyme: Molecular Basis of RNA binding and specificity by a AAA+ protein

Picornaviruses are one of the leading agents of animal and human infectious disease with at least 8 billion infections a year and cause a range of symptoms including respiratory failure and acute flaccid myelitis1. The most conserved nonstructural protein in picornaviruses is 2C2, a member of the AAA+ family of ATPases that binds RNA, and a broad spectrum antiviral target3-5. Despite its crucial role in the viral life cycle and as a clinical target, no structure of 2C bound to RNA has been structurally determined. Here we present the first structure of 2C as a hexamer bound to single stranded RNA in its central pore; a novel AAA+ protein:substrate interaction. Using the 2C:RNA holoenzyme complex structure, we characterize the mode that this AAA+ protein employs to specifically bind single stranded RNA, and demonstrate that mutations to key residues inhibit both RNA binding and viral replication in Apthovirus and Enterovirus systems, and show that the core residues responsible for binding are broadly conserved in viruses beyond Picornaviridae. Finally, we reveal that the 2C:RNA holoenzyme complex is conformationally more similar to a protein translocase adapted to bind RNA rather than other viral DNA binding SF3 helicases, underscoring how the AAA+ core module can be adapted for a variety of biochemical substrates.

biophysics↗

bioRxiv2026

Glio-SERS: Label-Free Molecular Profiling of Plasma Extracellular Vesicles in Brain Tumors Using SERS and Artificial Intelligence

Extracellular vesicles are increasingly recognized as important carriers of disease-associated molecular information, yet robust methods for their isolation and molecular characterization from limited clinical samples remain challenging. Here, we present an integrated approach combining standardized EV isolation, label-free Surface-Enhanced Raman Spectroscopy (SERS), and artificial intelligence (AI) for comprehensive molecular profiling of small extracellular vesicles (sEVs) from human plasma. Here, we show systematically isolated and characterized plasma sEVs using ExoTIC in accordance with MISEV2023 guidelines, with SERS analysis revealing quantifiable spectral differences across samples from patients with glioblastoma (n=20) and meningioma (n=23) compared to healthy controls (n=30). Among the evaluated AI models, the convolutional neural network most effectively captured group-level spectral differences in sEVs, achieving accuracies up to 88% in this pilot cohort. Further, an EGFR-based spectral regression model was explored to examine molecular variability across sEV samples. Parallel proteomic analysis presented statistically significant differences in several proteins elevated in glioblastoma or meningioma. This label-free, rapid approach provides a proof-of-concept framework for sEV molecular profiling establishing the basis for broad validation studies across diverse diseases.

bioengineering↗

bioRxiv2026

The ER membrane protein complex acts as a chaperone to promote the biogenesis of multi-bundle membrane proteins

Nearly half of the [~]5,000 human membrane proteins need to assemble into stoichiometric complexes as part of their biogenesis at the endoplasmic reticulum (ER) membrane. How ER resident biogenesis factors coordinate membrane insertion, folding and assembly is not well understood. Here, we demonstrate that the ER membrane protein complex (EMC) insertase additionally acts as a chaperone to facilitate the assembly of heterotrimeric voltage-gated calcium channels (Cav). Using function-separating mutations and inhibitory nanobodies we show that nascent Cav channels are degraded prematurely when EMCs chaperone function is selectively perturbed. Blocking EMCs chaperone function strongly impaired Cav-dependent cardiomyocyte contraction. EMC engagement of the pore-forming Cav-subunit occurred co-translationally and required Cavs first transmembrane domain bundle to protrude from the nascent ribosome*Sec61*multipass translocon complex. Our findings establish a chaperone function for the EMC and reveal that biogenesis of multi-bundle membrane proteins requires a highly orchestrated, co-translational interplay between ER biogenesis factors.

cell biology↗

bioRxiv2025

The Adhesion GPCR ADGRL2 engages Gα13 to Enable Epidermal Differentiation

Homeostasis relies on signaling networks controlled by cell membrane receptors. Although G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors, their specific roles in the epidermis are not fully understood. Dual CRISPR-Flow and single cell Perturb-seq knockout screens of all epidermal GPCRs were thus performed, uncovering an essential requirement for adhesion GPCR ADGRL2 (latrophilin 2) in epidermal differentiation. Among potential downstream guanine nucleotide-binding G proteins, ADGRL2 selectively activated G13. Perturb-seq of epidermal G proteins and follow-up tissue knockouts verified that G13 is also required for epidermal differentiation. A cryo-electron microscopy (cryo-EM) structure in lipid nanodiscs showed that ADGRL2 engages with G13 at multiple interfaces, including via a novel interaction between ADGRL2 intracellular loop 3 (ICL3) and a G13-specific QQQ glutamine triplet sequence in its GTPase domain. In situ gene mutation of this interface sequence impaired epidermal differentiation, highlighting an essential new role for an ADGRL2-G13 axis in epidermal differentiation.

cell biology↗

bioRxiv2024

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts

SNARE proteins drive membrane fusion at different cell compartments as their core domains zipper into a parallel four-helix bundle. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/-SNAP to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains. Previous structures of the NSF--SNAP-SNARE complex revealed binding to the D1 ATPase pore, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems. Using mass-spectrometry in yeast cells, we show N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. We present cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition, which show SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.

biophysics↗

bioRxiv2024

A synthetic cell-free pathway for biocatalytic upgrading of one-carbon substrates

Biotechnological processes hold tremendous potential for the efficient and sustainable conversion of one-carbon (C1) substrates into complex multi-carbon products. However, the development of robust and versatile biocatalytic systems for this purpose remains a significant challenge. In this study, we report a hybrid electrochemical-biochemical cell-free system for the conversion of C1 substrates into the universal biological building block acetyl-CoA. The synthetic reductive formate pathway (ReForm) consists of five core enzymes catalyzing non-natural reactions that were established through a cell-free enzyme engineering platform. We demonstrate that ReForm works in a plug-and-play manner to accept diverse C1 substrates including CO2 equivalents. We anticipate that ReForm will facilitate efforts to build and improve synthetic C1 utilization pathways for a formate-based bioeconomy.

synthetic biology↗

bioRxiv2024

Cryo-EM structures of the tubulin cofactors reveal the molecular basis for the biogenesis of alpha/beta-tubulin

Microtubule polarity and dynamic polymerization originate from the self-association properties of the a-tubulin heterodimer. For decades, it has remained poorly understood how the tubulin cofactors, TBCD, TBCE, TBCC, and the Arl2 GTPase mediate a-tubulin biogenesis from - and {beta}-tubulins. Here, we use cryogenic electron microscopy to determine structures of tubulin cofactors bound to {beta}-tubulin. These structures show that TBCD, TBCE, and Arl2 form a heterotrimeric cage-like TBC-DEG assembly around the a-tubulin heterodimer. TBCD wraps around Arl2 and almost entirely encircles -tubulin, while TBCE forms a lever arm that anchors along the other end of TBCD and rotates -tubulin. Structures of the TBC-DEG-{beta}-tubulin assemblies bound to TBCC reveal the clockwise rotation of the TBCE lever that twists a-tubulin by pulling its C-terminal tail while TBCD holds -tubulin in place. Altogether, these structures uncover transition states in {beta}-tubulin biogenesis, suggesting a vise-like mechanism for the GTP-hydrolysis dependent a-tubulin biogenesis mediated by TBC-DEG and TBCC. These structures provide the first evidence of the critical functions of the tubulin cofactors as enzymes that regulate the invariant organization of {beta}-tubulin, by catalyzing - and {beta}-tubulin assembly, disassembly, and subunit exchange which are crucial for regulating the polymerization capacities of {beta}-tubulins into microtubules.

biochemistry↗