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Semisynthesis of segmentally isotope-labeled and site-specifically palmitoylated CD44 cytoplasmic tail
Dominik P Vogl1, Borja Mateos2, Mario Migotti3
1University of Vienna, Faculty of Chemistry, Institute of Biological Chemistry, Währinger Str. 38, 1090 Vienna, Austria; University of Vienna, Vienna Doctoral School in Chemistry (DoSChem), Währinger Str. 42, 1090 Vienna, Austria.
Abstract:
CD44, a ubiquitously expressed transmembrane receptor, plays a crucial role in cell growth, migration, and tumor progression. Dimerization of CD44 is a key event in signal transduction and has emerged as a potential target for anti-tumor therapies. Palmitoylation, a posttranslational modification, disrupts CD44 dimerization and promotes CD44 accumulation in ordered membrane domains. However, the effects of palmitoylation on the structure and dynamics of CD44 at atomic resolution remain poorly understood. Here, we present a semisynthetic approach combining solid-phase peptide synthesis, recombinant expression, and native chemical ligation to investigate the impact of palmitoylation on the cytoplasmic domain (residues 669-742) of CD44 (CD44ct) by NMR spectroscopy. A segmentally isotope-labeled and site-specifically palmitoylated CD44 variant enabled NMR studies, which revealed chemical shift perturbations and indicated local and long-range conformational changes induced by palmitoylation. The long-range effects suggest altered intramolecular interactions and potential modulation of membrane association patterns. Semisynthetic, palmitoylated CD44ct serves as the basis for studying CD44 clustering, conformational changes, and localization within lipid rafts, and could be used to investigate its role as a tumor suppressor and to explore its therapeutic potential.
Insights
Palmitoylation, a modification of CD44 (a protein involved in tumor progression), alters its structure and dynamics. This finding provides a basis for exploring CD44
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- CD44 is a transmembrane receptor critical for cell growth, migration, and tumor progression.
- CD44 dimerization is vital for signal transduction and a target for anti-tumor therapies.
- Palmitoylation disrupts CD44 dimerization and influences its membrane localization, but its atomic-level effects are unclear.
Purpose of the Study:
- To investigate the impact of palmitoylation on the structure and dynamics of the CD44 cytoplasmic domain (CD44ct) at atomic resolution.
- To utilize a semisynthetic approach for creating site-specifically palmitoylated CD44 variants for NMR studies.
- To understand how palmitoylation affects CD44's conformational changes, intramolecular interactions, and membrane association.
Main Methods:
- Semisynthesis combining solid-phase peptide synthesis, recombinant expression, and native chemical ligation.
- NMR spectroscopy on segmentally isotope-labeled and site-specifically palmitoylated CD44 variants.
- Analysis of chemical shift perturbations to identify conformational changes.
Main Results:
- Palmitoylation induces local and long-range conformational changes in CD44ct, as evidenced by NMR chemical shift perturbations.
- Long-range effects suggest altered intramolecular interactions within CD44.
- Palmitoylation may modulate CD44's association patterns with cell membranes.
Conclusions:
- Semisynthetic palmitoylated CD44ct is a valuable tool for studying CD44 structure, dynamics, and function.
- Palmitoylation-induced conformational changes may influence CD44 clustering and localization in lipid rafts.
- This research opens avenues for investigating CD44's tumor suppressor role and therapeutic potential.
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