| Autor: |
Simon DN; Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. dsimon01@rockefeller.edu., Wriston A; Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA. asw5kg@virginia.edu., Fan Q; Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway. qiong.fan@medisin.uio.no., Shabanowitz J; Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA. js4c@virginia.edu., Florwick A; Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. alyssa.florwick@duke.edu., Dharmaraj T; Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. tdharma1@jhu.edu., Peterson SB; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. speter54@its.jnj.com., Gruenbaum Y; Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram Jerusalem 91904, Israel. gru@mail.huji.ac.il., Carlson CR; Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway. c.r.carlson@medisin.uio.no., Grønning-Wang LM; Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway. lmgronningwang@gmail.com., Hunt DF; Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA. dfh@virginia.edu.; Department of Pathology, University of Virginia, Charlottesville, VA 22904, USA. dfh@virginia.edu., Wilson KL; Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. klwilson@jhmi.edu. |
| Abstrakt: |
The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases ('laminopathies'). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β- O -linked N -acetylglucosamine- (O -GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O -GlcNAc transferase (OGT) enzyme showed robust O -GlcNAcylation of recombinant mature lamin A tails (residues 385⁻646), with no detectable modification of lamin B1, lamin C, or 'progerin' (Δ50) tails. Using mass spectrometry, we identified 11 O -GlcNAc sites in a 'sweet spot' unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O -GlcNAc-modified at seven sites. By contrast, O -GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson⁻Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O -GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622⁻625 and 639⁻645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A. |
