Phase‑Separation‑Driven Sequestration of DDR Kinases by Farnesylated Prelamin A Links ZMPSTE24 Decline to Age‑Related Genomic Instability

Hypothesis

Age‑related decline of ZMPSTE24 leads to accumulation of farnesylated prelamin A that drives aberrant phase separation of the nuclear lamina, creating microdomains that sequester multiple DDR kinases (CK2α, ATM, DNA‑PKcs) and suppress their activity, thereby causing replication stress and R‑loop‑mediated genome instability.

Mechanistic Rationale

1. Farnesylated prelamin A is more hydrophobic and prone to self‑assembly, a property known to promote liquid‑liquid phase separation (LLPS) of nuclear envelope proteins.
2. The lamina already functions as a signaling scaffold that tethers kinases; LLPS would increase the local concentration and residency time of these kinases within prelamin A‑rich condensates, reducing their diffusion‑dependent access to chromatin lesions.
3. In progeria, mutant prelamin A forms stable CK2α complexes; we extend this to physiological aging where modest prelamin A elevation still reaches a threshold for LLPS‑mediated kinase trapping.
4. Sequestered kinases fail to phosphorylate downstream substrates (e.g., H2AX, 53BP1, RPA), impairing DDR signaling, fork restart, and R‑loop resolution, which explains the overlap of replication stress and R‑loop accumulation observed in aged tissues.
5. Spermidine rescues progeria by disrupting prelamin A‑CK2α binding; we propose it also interferes with the multivalent interactions that underlie prelamin A LLPS, dissolving condensates and restoring kinase mobility.

Testable Predictions

• Prediction 1: In nuclei from aged human arteries or Lmna L648R/L648R mouse tissues, farnesylated prelamin A will colocalize with CK2α, ATM, and DNA‑PKcs in distinct micron‑sized foci that exhibit LLPS characteristics (sensitivity to 1,6‑hexanediol, rapid FRAP recovery).
• Prediction 2: Acute depletion of ZMPSTE24 in young cells will increase prelamin A‑dependent condensate formation, reduce kinase chromatin binding, and elevate γH2AX and R‑loop levels; these effects will be reversed by spermidine or by a farnesyltransferase inhibitor.
• Prediction 3: Genetic disruption of prelamin A’s farnesylation site (C661S) or expression of a non‑farnesylated prelamin A mutant will prevent condensate formation despite ZMPSTE24 loss, preserving DDR signaling and reducing genomic instability markers.
• Prediction 4: Overexpressing a soluble CK2α mutant that lacks the lamin‑binding domain will rescue DDR activity in aged cells without altering prelamin A levels, indicating that kinase sequestration—not merely reduced expression—is pathogenic.

Experimental Approach

1. Imaging & Biophysics: Perform immunofluorescence for prelamin A, CK2α, ATM, and DNA‑PKcs in aortic sections from young vs. aged mice and human donors. Apply 1,6‑hexanediol treatment to test LLPS sensitivity and conduct FRAP on GFP‑tagged kinases to measure mobility.
2. Biochemical Fractionation: Isolate chromatin‑bound vs. soluble kinase fractions from nuclear extracts; quantify by western blot and mass spectrometry to detect shifts toward the soluble fraction upon spermidine or farnesyltransferase inhibition.
3. Functional Assays: Measure DNA fiber assay replication fork speed, R‑loop levels (S9.6 immunofluorescence), and γH2AX foci after inducing replication stress (hydroxyurea) or transcription inhibition (DRB).
4. Genetic Manipulations: Use CRISPR to introduce C661S Lmna or Lmna‑null backgrounds in cultured vascular smooth muscle cells; assess whether these manipulations block condensate formation and rescue DDR phenotypes under ZMPSTE24 knockdown.
5. In Vivo Validation: Treat aged Lmna L648R/L648R mice with spermidine or a farnesyltransferase inhibitor (lonafarnib) and evaluate nuclear morphology, kinase localization, and markers of genomic instability in aorta and heart.

If these experiments confirm that farnesylated prelamin A drives LLPS‑mediated kinase sequestration, it will establish a unifying mechanism linking ZMPSTE24 decline to age‑related genome instability and reveal new therapeutic angles targeting lamina phase behavior rather than protein levels alone.