Biology subjects

Dharan, A.

Publications and source records attributed to Dharan, A..

3 recordsLinked to original sources

bioRxiv2026

Stereochemical identity of lipid nanoparticles modulates protein expression via internal lipid organization

Stereochemistry plays a crucial role in how molecules interact with complex physiological environments, affecting pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Although these effects are well studied for small-molecular drugs, they are largely overlooked for supramolecular assemblies used in drug delivery. Even for lipid nanoparticles (LNPs)--the most advanced RNA delivery platform--stereochemical effects are rarely investigated and, when considered, are typically limited to the ionizable lipid rather than the overall stereochemical identity of the LNP. Here we separate the ionizable lipid cKK-E12 into its two stereoisomers (trans: R,S/S,R; cis: R,R/S,S), which are normally used as a mixture. LNPs containing the cis isomer exhibit improved physicochemical properties, stability, and protein expression. By systematically varying the stereochemistry of the ionizable lipid, phospholipid, and cholesterol, we reveal stereochemistry-dependent differences in uptake and protein expression across six cell lines and in vivo in zebrafish embryos and mice. AI-assisted cryo-TEM analysis and SAXS link enhanced protein expression to structural differences, demonstrating control over internal lipid phases (lamellar and inverse hexagonal), influencing sample uniformity, and identifying stereochemical identity as a key determinant of functional RNA delivery.

pharmacology and toxicology↗

bioRxiv2024

The nuclear localization signal of CPSF6 governs post-nuclear import steps of HIV-1 infection

The early stages of HIV-1 infection include the trafficking of the viral core into the nucleus of infected cells. However, much remains to be understood about how HIV-1 accomplishes nuclear import and the consequences of the import pathways utilized on nuclear events. The host factor cleavage and polyadenylation specificity factor 6 (CPSF6) assists HIV-1 nuclear localization and post-entry integration targeting. Here, we used a CPSF6 truncation mutant lacking a functional nuclear localization signal (NLS), CPSF6-358, and appended heterologous NLSs to rescue nuclear localization. We show that some, but not all, NLSs drive CPSF6-358 into the nucleus. Interestingly, we found that some nuclear localized CPSF6-NLS chimeras supported inefficient HIV-1 infection. We found that HIV-1 still enters the nucleus in these cell lines but fails to traffic to speckle-associated domains (SPADs). Additionally, we show that HIV-1 fails to efficiently integrate in these cell lines. Collectively, our results demonstrate that the NLS of CPSF6 facilitates steps of HIV-1 infection subsequent to nuclear import and additionally identify the ability of canonical NLS sequences to influence cargo localization in the nucleus following nuclear import. Author SummaryDuring HIV-1 infection, the viral capsid, which encloses the viral genome and accessory proteins required for reverse transcription (RT) and integration, traffics towards the nucleus and enters through the nuclear pore complex (NPC). Following entry into the nucleus, RT is completed and viral capsid disassembles releasing the preintegration complex (PIC) to integrate with the host chromosome. In this study, we investigated the early HIV-1 host factor CPSF6, and specifically focused on the C-terminal short amino acid nuclear localization signal (NLS) in CPSF6, in mediating viral nuclear entry and subsequent gene expression. Altering the NLS in CPSF6 with NLS from other proteins, significantly impacted HIV-1s ability to infect those cells. We further showed this defect in infection occurred at the level of viral integration. This study highlights the importance of the NLS in CPSF6 in dictating the NPC it associates with and its effect on HIV-1 infection. Moreover, our study emphasizes the function of NLS in targeting host cargos to different nuclear entry pathways.

cancer biology↗

bioRxiv2024

Arc mediates intercellular synaptic plasticity via IRSp53-dependent extracellular vesicle biogenesis.

Current models of learning and memory have focused on cell-autonomous regulation of synaptic strength; however, intercellular signaling between cells in the brain is important for normal cognition. The immediate early gene Arc is a repurposed retrotransposon critical for long-term forms of synaptic plasticity and memory. Arc protein forms virus-like capsids released in extracellular vesicles (EVs) that mediate intercellular signaling of unknown function. Here, we find that long-term potentiation stimuli induce the biogenesis of Arc EVs by recruiting the I-BAR protein IRSp53, which facilitates Arc capsid assembly, trafficking, and release from actin-rich filopodial structures in dendrites. Arc EVs transfer Arc protein and mRNA to neighboring dendrites, where translation of transferred Arc mRNA induces a loss of surface AMPA-type glutamate receptors. These results show that Arc EVs mediate an intercellular form of synaptic plasticity that may be critical for memory consolidation and reveals a new neuronal EV biogenesis pathway.

neuroscience↗