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The activation of transcription factor Max-Like Protein x (MLX) is modulated by competition between active dimerization and inactive association with cytosolic lipid droplets (LDs). However, LD association has been shown to depend on the neutral lipid composition. This work explores the mechanism by which MLX specifically targets LDs rich in triacylglycerol (TG) over those with abundant sterol esters (SE). We compare the association ensembles for a potential minimal targeting sequence, an amphipathic helix-loop-helix hairpin, and the full dimerization and cytoplasmic localization domain (DCD), finding the latter requires larger packing defects and quantifiably alters LD membrane properties. Surprisingly, direct interactions with TG neutral lipids are not observed for either sequence. Instead, targeting to SE-rich LDs is blocked for both sequences by insufficient packing defects. We additionally explore the full mechanism of hairpin association, aiming to understand sequence-specific features that enable strong membrane association. We find that there are multiple association pathways, but that each involves a catch, dive, snorkeling, and embedding phase. The combination of multiple catch and dive residues placed on opposing ends of amphipathic helices lengthens the catch phase, greatly enhancing association in a manner that resembles kinetic selection. Once bound, locking interhelical interactions block dissociation. Collectively, our findings suggest that in addition to relative binding affinities, both kinetics and altered surface properties due to protein association could influence competition within the LD proteome. SignificanceThe transcription factor Max-Like Protein x (MLX plays) a central role in metabolic regulation by responding to nutrient status and, simultaneously, neutral lipid composition. This work reveals how MLX selectively targets triacylglycerol-rich lipid droplets (LDs) through sequence-specific interactions with packing defects. We show that LD surface modulates MLX binding and that MLX in turn alters monolayer properties, highlighting a dynamic interplay between protein association and membrane properties. These findings provide new insight into how protein localization and function may be regulated at LD surfaces, with implications for nutrient sensing and, more broadly, transcriptional control relevant to metabolic and disease states.
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The C-Terminus of Perilipin 3 Shows Distinct Lipid Binding at Phospholipid-Oil-Aqueous Interfaces
Lipid droplets (LDs) are ubiquitously expressed organelles; the only intracellular organelles that contain a lipid monolayer rather than a bilayer. Proteins localize and bind to this monolayer as they do to intracellular lipid bilayers. The mechanism by which cytosolic LD binding proteins recognize, and bind, to this lipid interface remains poorly understood. Amphipathic α-helix bundles form a common motif that is shared between cytosolic LD binding proteins (e.g., perilipins 2, 3, and 5) and apolipoproteins, such as apoE and apoLp-III, found on lipoprotein particles. Here, we use pendant drop tensiometry to expand our previous work on the C-terminal α-helix bundle of perilipin 3 and the full-length protein. We measure the recruitment and insertion of perilipin 3 at mixed lipid monolayers at an aqueous-phospholipid-oil interface. We find that, compared to its C-terminus alone, the full-length perilipin 3 has a higher affinity for both a neat oil/aqueous interface and a phosphatidylcholine (PC) coated oil/aqueous interface. Both the full-length protein and the C-terminus show significantly more insertion into a fully unsaturated PC monolayer, contrary to our previous results at the air-aqueous interface. Additionally, the C-terminus shows a preference for lipid monolayers containing phosphatidylethanolamine (PE), whereas the full-length protein does not. These results strongly support a model whereby both the N-terminal 11-mer repeat region and C-terminal amphipathic α-helix bundle domains of perilipin 3 have distinct lipid binding, and potentially biological roles.
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- Award ID(s):
- 1808281
- PAR ID:
- 10297280
- Date Published:
- Journal Name:
- Membranes
- Volume:
- 11
- Issue:
- 4
- ISSN:
- 2077-0375
- Page Range / eLocation ID:
- 265
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/membranes11040265
