Smurf2 regulates stability and the autophagic–lysosomal turnover of lamin A and its disease‐associated form progerin

Summary A‐lamins, encoded by the LMNA gene, are major structural components of the nuclear lamina coordinating essential cellular processes. Mutations in the LMNA gene and/or alterations in its expression levels have been linked to a distinct subset of human disorders, collectively known as laminopathies, and to cancer. Mechanisms regulating A‐lamins are mostly obscure. Here, we identified E3 ubiquitin ligase Smurf2 as a physiological regulator of lamin A and its disease‐associated mutant form progerin (LAΔ50), whose expression underlies the development of Hutchinson‐Gilford progeria syndrome (HGPS), a devastating premature aging syndrome. We show that Smurf2 directly binds, ubiquitinates, and negatively regulates the expression of lamin A and progerin in Smurf2 dose‐ and E3 ligase‐dependent manners. Overexpression of catalytically active Smurf2 promotes the autophagic–lysosomal breakdown of lamin A and progerin, whereas Smurf2 depletion increases lamin A levels. Remarkably, acute overexpression of Smurf2 in progeria fibroblasts was able to significantly reduce the nuclear deformability. Furthermore, we demonstrate that the reciprocal relationship between Smurf2 and A‐lamins is preserved in different types of mouse and human normal and cancer tissues. These findings establish Smurf2 as an essential regulator of lamin A and progerin and lay a foundation for evaluating the efficiency of progerin clearance by Smurf2 in HGPS, and targeting of the Smurf2–lamin A axis in age‐related diseases such as cancer.


Cell transfections and generation of stable cell lines
cDNA transfections were performed using FUGENE 6 (Promega) according to the manufacturer's instructions. For siRNAs transfections, Oligofectamine (Invitrogen) was used. For Smurf2 overexpression in HGADFN167 and HGFDFN168 fibroblasts, cells were electroporated (Lonza VCA1001) with 5 µg of GFP-Smurf2 or GFP-empty vector, and analyzed 48 hrs after electroporation. For generating of Smurf2 stable knock-down, cells were infected with lentiviruses containing pLKO.1-Smurf2-puro vector (Sigma), and selected with puromycin for 2-3 weeks.

GST-fusion protein, pull-down assays and ubiquitination assays
GST fusion proteins were prepared from E.coli and isolated using Glutathione Sepharose 4B beads (GE Healthcare). Flag-lamin A and Flag-progerin were produced using TNT® SP6 Coupled Wheat Germ Extract System (L3260; Promega).
For the in vitro binding assay, Flag-tagged proteins were first pre-cleared with Glutathione-Sepharose beads for 1 hr at 4°C. After pre-clearing, these proteins were incubated with purified GST-Smurf2 or GST proteins in binding buffer. GST pull-down was conducted using Glutathione Sepharose 4B beads. Flag-progerin was pulled-down using agarose beads conjugated with FLAG antibody (FLAG-M2 affinity gel; Sigma-Aldrich). Beads were then washed four times with ice-cold binding buffer, and proteins were eluted with 5X SDS sample buffer.
In vivo and in vitro ubiquitination assays were performed as previously described (Levy-Cohen et al., 2015;Emanuelli et al., 2017). In brief, for the in vivo ubiquitination assay cells were lysed with RIPA buffer supplemented with 5 mM NEM (N-Ethylmaleimide). Flag-lamin A and Flag-progerin were immunoprecipitated, and their ubiquitination pattern analyzed.
For the in vitro ubiquitination assay, Flag-lamin A and Flag-progerin derived from the TNT® reaction were incubated with 2 µg of GST or GST-Smurf2, 5 µg of HA-ubiquitin protein, E1 (UBE1; 100 ng), E2 enzyme (UbcH5c; 150 ng), and 100 mM ATP-Mg in the E3 ligase reaction buffer (BostonBiochem) for 2 hrs at 37°C. RIPA buffer was added to the reactions and Flag-lamin A and Flag-progerin were pulled down using M2-FLAG beads (Sigma).

qRT-PCR
Total RNA was extracted from Smurf2 knock-down and control MDA-MB-231 cells using RNeasy mini kit (Qiagen), according to the manufacturer's instructions. Total RNA was then reversetranscribed with random primers using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). LMNA cDNA levels were determined using Fast SYBR Green Master mix and ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific). The experiments were performed three times with three technical replicates for each experiment. The gene expression was calculated using 2 -ΔΔCt method, and normalized to GAPDH gene. The following primers were used for lamin A/C expression analysis: Forward: 5'-aatgatcgcttggcggtctac-3' and Reverse: 5'-cttcttggtattgcgcgcttt-3'.

Nuclear circularity/deformability analysis
Nuclear circularity was measured as previously described (Goldman et al., 2004). Briefly, the circularity of the nucleus with a perfect circle shape was scored as 1.0. Nuclei with lobulation and/or blebbing were valued between >0 and <1.0, depending on the severity of the phenomena. The outline of the nuclei, obtained under confocal fluorescent microscope, was traced with a freehand selection tool, followed by the measurement of nuclear circularity with the ImageJ software (the software measures the roundness of the nucleus using the formula: 4π×area/perimeter 2 ).

Statistical analysis
Two-tailed student t-test was applied for statistical analysis of data. Data with P-values of less than 0.05 were considered statistically significant.    Figure S4. Smurf2 overexpression triggers lamin A association with the lysosomal protein LAMP1. HEK-293T cells co-expressing mCherry-lamin A and GFP-Smurf2 (or an empty GFP vector) were immunostained with anti-LAMP1 antibody, and the co-localization between these proteins in untreated and chloroquine-treated cells was visualized under confocal microscope. Left panel shows quantification of the confocal data obtained in two independent experiments with an average of 96 cells/group. Data are mean + SEM. **P < 0.01, ***P < 0.001. Representative confocal images, indicating the association sites of mCherry-lamin with LAMP1 in Smurf2-overexpressing cells, are shown on the right. Bars, 10 µm.