Bharti Singal

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.

SUMMARY: 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 (Ca _v ). Using function-separating mutations and inhibitory nanobodies we show that nascent Ca _v channels are degraded prematurely when EMC’s chaperone function is selectively perturbed. Blocking EMC’s chaperone function strongly impaired Ca _v -dependent cardiomyocyte contraction. EMC engagement of the pore-forming Ca _v α-subunit occurred co-translationally and required Ca _v α’s 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.

SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) 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+ (ATPase associated with diverse cellular activities) protein Sec18/NSF and its adaptor Sec17/α-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–α-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-electron microscopy (cryo-EM) structures of a yeast SNARE complex, Sec18 and Sec17 in a nonhydrolyzing 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.

One-fifth of human proteins are N-glycosylated in the endoplasmic reticulum (ER) by two oligosaccharyltransferases, OST-A and OST-B. Contrary to the prevailing view of N-glycosylation as a housekeeping function, we identified an ER pathway that modulates the activity of OST-A. Genetic analyses linked OST-A to HSP90B1, an ER chaperone for membrane receptors, and CCDC134, an ER luminal protein. During its translocation into the ER, an N-terminal peptide in HSP90B1 templates the assembly of a translocon complex containing CCDC134 and OST-A that protects HSP90B1 during folding, preventing its hyperglycosylation and degradation. Disruption of this pathway impairs WNT and IGF1R signaling and causes the bone developmental disorder osteogenesis imperfecta. Thus, N-glycosylation can be regulated by specificity factors in the ER to control cell surface receptor signaling and tissue development.

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 α- and β-tubulins. Here, we use cryogenic electron microscopy to determine structures of tubulin cofactors bound to αβ-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 α-tubulin. Structures of the TBC-DEG-αβ-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 αβ-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 αβ-tubulin, by catalyzing α- and β-tubulin assembly, disassembly, and subunit exchange which are crucial for regulating the polymerization capacities of αβ-tubulins into microtubules.

The stringent response, regulated by the bifunctional (p)ppGpp synthetase/hydrolase Rel in mycobacteria, is critical for long‐term survival of the drug‐tolerant dormant state of Mycobacterium tuberculosis. During amino acid starvation, MtRel senses a drop in amino acid concentration and synthesizes the messengers pppGpp and ppGpp, collectively called (p)ppGpp. Here, we investigate the role of the regulatory ‘Aspartokinase, Chorismate mutase and TyrA’ (ACT) domain in MtRel. Using NMR spectroscopy approaches, we report the high‐resolution structure of dimeric MtRel ACT which selectively binds to valine out of all other branched‐chain amino acids tested. A set of MtRel ACT mutants were generated to identify the residues required for maintaining the head‐to‐tail dimer. Through NMR titrations, we determined the crucial residues for binding of valine and show structural rearrangement of the MtRel ACT dimer in the presence of valine. This study suggests the direct involvement of amino acids in (p)ppGpp accumulation mediated by MtRel independent to interactions with stalled ribosomes.DatabaseStructural data are available in the PDB database under the accession number 6LXG.

Modulation of intracellular guanosine 3′,5′‐bispyrophosphate ((p)ppGpp) level, the effector of the stringent response, is crucial for survival as well as optimal growth of prokaryotes and, thus, for bacterial pathogenesis and dormancy. In Mycobacterium tuberculosis (Mtb), (p)ppGpp synthesis and degradation are carried out by the bifunctional enzyme MtRel, which consists of 738 residues, including an N‐terminal hydrolase‐ and synthetase‐domain (N‐terminal domain or NTD) and a C‐terminus with a ribosome‐binding site. Here, we present the first crystallographic structure of the enzymatically active MtRel NTD determined at 3.7 Å resolution. The structure provides insights into the residues of MtRel NTD responsible for nucleotide binding. Small‐angle X‐ray scattering experiments were performed to investigate the dimeric state of the MtRel NTD and possible substrate‐dependent structural alterations.

Neisseria meningitides is a parasitic gram-negative bacterium of the family Neisseriaceae (Proteobacteria) and it causes many human diseases including meningitidis and septicemia. One of its strains, H44/76, has natural transformation capacity, thus it is important to identify possible novel drug targets and to develop serogroup B vaccines against this opportunist pathogen. In the complete genome of N. meningitides strain H44/76, there are 1961 coding genes out of which 544 encodes for hypothetical proteins (HPs). Due to their less homology and relatedness to other known proteins, HPs may serve as potential drug targets. We performed extensive functional analysis of these HPs with the help of Bioinformatics tools and assigned functions to 235 HPs, out of which 202 were annotated with high confidence whereas 33 with less confidence. In this study, we have used a combination of latest tools to acquire information about the conserved regions, families, pathways, interactions, localization and virulence related to a particular protein. We also categorized these proteins as transporters, regulators, enzymes, binding proteins, virulent proteins. The outcome of this intensive study may help in the comprehensive understanding of pathogenesis, drug resistance, adaptability to host, epidemic causes and drug discovery for treatment of the diseases.