Biology subjects

Nag, M.

Publications and source records attributed to Nag, M..

3 recordsLinked to original sources

bioRxiv2024

Ex Vivo Delivery of mRNA to Immune Cells Via a Non-Endosomal Route Obviates the Need for Nucleoside Modification

Base modification and the use of lipid nanoparticles (LNPs) are thought to be essential for efficient in vivo delivery and expression of mRNA. However, for ex vivo immune cell engineering, the need for either of the two is unclear. Previous reports have suggested that nucleic acids may be efficiently delivered to immune cells ex vivo, through a non-endosomal delivery route, but the need for base modification has not been determined. Herein, we demonstrate that when a non-endosomal delivery method is used, unmodified mRNA performs equally well to the commonly used base-modified mRNA, including the N1 methyl pseudo-uridine modification, in terms of protein expression and inflammatory response in cells. However, if an endosomal delivery route is used, then N1 methyl pseudo-uridine modification is necessary for high expression and low inflammatory response, as demonstrated by others as well. Overall, we show that non-endosomal mRNA delivery renders nucleoside modifications non-essential, and that unmodified mRNA combined with non-endosomal delivery route may be used for efficient ex vivo mRNA-based engineering of immune cells.

bioengineering↗

bioRxiv2024

Structural insights into IMP2 dimerization and RNA binding

IGF2BP2 (IMP2) is an RNA-binding protein that contributes to cancer tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; however, structural information on full-length IMP2 is lacking but necessary to understand how to target IMP2 activity in drug discovery. In this study, we investigated the behavior of full-length IMP2 and the influence of RNA binding using biophysical and structural methods including mass photometry, hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), and small angle x-ray scattering (SAXS). We found that full-length IMP2 forms multiple oligomeric states but predominantly adopts a dimeric conformation. Molecular models derived from SAXS data suggest the dimer is formed in a head-to-tail orientation by the KH34 and RRM1 domains. Upon RNA binding, IMP2 forms a pseudo-symmetric dimer different from its apo/RNA-free state, with the KH12 domains of each IMP2 molecule forming the dimer interface. We also found that the formation of IMP2 oligomeric species, which includes dimers and higher-order oligomers, is sensitive to ionic strength and RNA binding. Our findings provide the first insight into the structural properties of full-length IMP2, which may lead to novel opportunities for disrupting its function with more effective IMP2 inhibitors.

molecular biology↗

bioRxiv2023

Conserved folding landscape of monomeric initiator caspases

The apoptotic caspase subfamily evolved into two subfamilies - monomeric initiators and dimeric effectors. Sequence variations in the conserved caspase-hemoglobinase fold resulted in changes in oligomerization, enzyme specificity, and regulation, making caspases an excellent model for examining the mechanisms of molecular evolution in fine-tuning structure, function, and allosteric regulation. We examined the urea-induced equilibrium folding/unfolding of two initiator caspases, monomeric caspase-8 and cFLIPL, over a broad pH range. Both proteins unfold by a three-state equilibrium mechanism that includes a partially folded intermediate. In addition, both proteins undergo a conserved pH-dependent conformational change that is controlled by an evolutionarily conserved mechanism. We show that the conformational free energy landscape of the caspase monomer is conserved in the monomeric and dimeric subfamilies. Molecular dynamics simulations in the presence or absence of urea, coupled with limited trypsin proteolysis and mass spectrometry, show that the small subunit is unstable in the protomer and unfolds prior to the large subunit. In addition, the unfolding of helix 2 in the large subunit results in disruption of a conserved allosteric site. Because the small subunit forms the interface for dimerization, our results highlight an important driving force for the evolution of the dimeric caspase subfamily through stabilizing the small subunit.

biophysics↗