Biology subjects

Shahzad, S.

Publications and source records attributed to Shahzad, S..

4 recordsLinked to original sources

bioRxiv2026

Lipid nanoparticle shape and morphology determine the magnitude and duration of mRNA transfection

Lipid nanoparticles (LNP) are widely used for nucleic acid delivery, with current optimization efforts mainly focusing on refining formulation features, enabling extrahepatic targeting, and improving therapeutic efficacy. Particle shape and internal architecture are variables often overlooked in LNP design, although they critically influence particle stability, biodistribution, cellular uptake, and transfection. Here, we investigate how formulation and process parameters impact LNP shape, morphology, and internal structure, and how these structural features, in turn, influence mRNA loading, cell uptake, and expression. We demonstrate that modulation of lipid composition by increasing DSPC content drives a transition from compact spherical to vesicular non-spherical morphologies, with altered RNA loading at the single-particle level. While non-spherical LNP exhibited higher cellular uptake than spherical LNP, it was identified that this increase was relatively modest compared with the substantially higher and sustained protein expression for non-spherical structures. This suggests that increased uptake alone cannot explain the enhanced transfection performance. The trend was conserved across LNP formulations comprising clinically relevant ionizable lipids SM-102, ALC-0315 and DLin-MC3-DMA. Taken together, these findings establish LNP shape and internal morphology as functionally relevant design variables that should be considered in the design of next-generation LNP with improved mRNA delivery and expression.

pharmacology and toxicology↗

bioRxiv2024

TDP43 autoregulation gives rise to shortened isoforms that are tightly controlled by both transcriptional and post-translational mechanisms

The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, highly prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a byproduct of TDP43 autoregulation and cleared by nonsense mediated RNA decay (NMD). The sTDP43-encoding transcripts that escape NMD can lead to toxicity but are rapidly degraded post-translationally. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration in vitro and in vivo via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and provide insight into the consequences of aberrant sTDP43 accumulation in disease.

neuroscience↗

bioRxiv2023

Optical ice thickness measurement in the VitroJet for time-efficient single particle structure determination

Embedding biomolecules in vitreous ice of optimal thickness is critical for structure determination by cryo-electron microscopy. Ice thickness assessment and selection of suitable holes for data collection are currently part of time-consuming preparatory routines performed on expensive electron microscopes. To address this challenge, a routine has been developed to measure ice thickness during sample preparation using an optical camera integrated in the VitroJet. This method allows to estimate the ice thickness with an error below {+/-} 20 nm for ice layers in the range of 0 - 70 nm. Additionally, we implemented pin printing to reproduce and control sample deposition in the VitroJet. The median ice thickness can be reproduced with a standard deviation below {+/-} 11 nm for thicknesses up to 75 nm. Therefore, the ice thickness of buffer-suspended holes on an EM grid can be tuned and measured within the working range relevant for single particle cryo-EM. Single particle structures of apoferritin were determined at two distinct thicknesses of 30 nm and 70 nm. These reconstructions demonstrate the importance of ice thickness for time-efficient cryo-EM structure determination. HighlightsO_LIMethods in the VitroJet allow for on-the-fly ice thickness tuning and measurement C_LIO_LIThe optical camera can estimate ice thickness ranging from 0 - 70 {+/-} 20 nm C_LIO_LIPin printing enables to reproduce and control median ice thickness up to 75 {+/-} 11 nm C_LIO_LIStructures of apoferritin require 3.7 x fewer particles in 30 compared to 70 nm ice C_LI

molecular biology↗

bioRxiv2021

A dual allosteric pathway drives human mitochondrial Lon

The hexameric, barrel-forming, AAA+ protease Lon is critical for maintaining mitochondrial matrix protein homeostasis. Efficient substrate processing by Lon requires the coordinated action of six protomers. Despite Lons importance for human health, the molecular bases for Lons substrate recognition and processing remain unclear. Here, we use a combination of biochemistry and electron cryomicroscopy (cryo-EM) to unveil the structural and functional basis for full-length human mitochondrial Lons degradation of mitochondrial transcription factor A (TFAM). We show how opposing protomers in the Lon hexamer barrel interact through their N-terminal domains to give what resembles three feet above the barrel and help to form a triangular pore located just above the entry pore to the barrel. The interactions between opposing protomers constitute a primary allosteric regulation of Lon activity. A secondary allosteric regulation consists of an inter-subunit signaling element in the ATPase domains. By considering the ATP or ADP load in each protomer, we show how this dual allosteric mechanism in Lon achieves coordinated ATP hydrolysis and substrate processing. This mechanism enforces sequential anti-clockwise ATP hydrolysis resulting in a coordinated hand-over-hand translocation of the substrate towards the protease active sites.

biochemistry↗