Biology subjects

Doutch, J. J.

Publications and source records attributed to Doutch, J. J..

2 recordsLinked to original sources

bioRxiv2025

Insight into the structure and interactions of the M. tuberculosis Mce-associated membrane proteins Mam1A-1D

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to more than a million human deaths every year. Mammalian cell entry complexes (Mce1-4) play an essential role in the survival of Mtb during the latent stage by mediating the import of lipids, including fatty acids and cholesterol, from the host. The proper functioning of Mce-complexes requires additional proteins such as Mce-associated membrane (Mam) proteins and lipid uptake coordinator (LucA), thus making them potential candidates for the development of anti-TB drugs. Four Mam (Mam1A-1D) proteins are coded from the mce1 operon and two from the mce3 (Mam3A-3B) and mce4 (Mam4A-4B) operons. In addition, five orphaned mam (Omam) proteins have been identified, which are not part of the mce operons but are functionally relevant for the Mce complexes. Analysis of the sequences of Mam/Omam proteins suggests that they share many common secondary and tertiary structural elements despite the low sequence identity between them. Here, we have characterized a recombinantly produced Mam1A variant by small-angle X-ray and neutron scattering. The studies indicate that Mam1A is tetrameric in solution with two disulfide bridges necessary for the stability of Mam1A. Similarly, a disulfide bridge has also been identified in Mam1C. Furthermore, through coexpression and copurification, we demonstrate that Mam1A-1D and LucA interact to form stable Mam1ABCD as well as Mam1ABCD-LucA complexes. The results obtained pave the way for further understanding how the Mam1ABCD and Mam1ABCD-LucA complexes are organized and interact with the Mce complexes, leading to their stabilization.

biochemistry↗

bioRxiv2021

Time evolution of PEG-shedding and serum protein coronation determines the cell uptake kinetics and delivery of lipid nanoparticle formulated mRNA

Development of efficient lipid nanoparticle (LNP) vectors remains a major challenge towards broad clinical translation of RNA therapeutics. New lipids will be required, but also better understanding LNP interactions with the biological environment. Herein, we model protein corona formation on PEG-ylated DLin-MC3-DMA LNPs and identify time-dependent maturation steps that critically unlock their cellular uptake and mRNA delivery. Uptake requires active serum proteins and precedes after a significant ([~]2 hours) lag-time, which we show can be eliminated by pre-incubating LNPs for 3-4 hours in serum-containing media. This indicates an important role of protein corona maturation for the pharmacokinetic effects of these LNPs. We show, using single-nanoparticle imaging, NMR diffusometry, SANS, and proteomics, that the LNPs, upon serum exposure, undergo rapid PEG-shedding ([~]30 minutes), followed by a slower rearrangement of the adsorbed protein layer. The PEG-shedding coincides in time with high surface abundance of Apolipoprotein A-II, whereas the LNPs preferentially bind Apolipoprotein E when their maximum uptake-competent state is reached. Finally, we show that pre-incubation of the LNPs enables rapid uptake and allows pulse-chase video-microscopy colocalization experiments with sufficiently short pulse durations to gain improved mechanistic understanding of how intracellular trafficking events determine delivery efficacy, emphasizing early endosomes as important delivery-mediating compartments.

biophysics↗