Abstract 4144933: Telomeric Repeat-Binding Factor 2 Interacting Protein (TERF2IP) S205 Phosphorylation Orchestrates LATS1/2 Degradation under Disturbed Flow Conditions

Background: While the shelterin complex, including TERF2IP, is established in its role of telomere protection, its interaction with the Hippo pathway, which also regulates senescence and inflammation, has not been reported. Our research aims to investigate whether TERF2IP influences the Hippo pathway, potentially linking it to endothelial cell (EC) responses under disturbed flow (d-flow). Methods: ECs were transduced with TERF2IP variants, and subjected to d-flow or static conditions. Post-transfection with makron-1 ubiquitin E3 ligase (MKRN1)/control siRNA, ECs underwent d-flow or remained static. Protein turnover was analyzed using cycloheximide (CHX) treatment. TERF2IP-MKRN1/LATS1/2 interactions were assessed via co-immunoprecipitation after d-flow exposure. Results: LATS1/2 depletion led to endothelial cell (EC) senescence and apoptosis, prompting investigation into the regulatory role of TERF2IP S205 phosphorylation on d-flow-induced LATS1/2 degradation. Adenoviral transduction with the TERF2IP S205A mutation significantly mitigated the d-flow-mediated decrease in LATS1/2 levels. The absence of changes in LATS1/2 mRNA post-d-flow and the protective effect of the protease inhibitor MG132 against LATS1/2 reduction implicate protein degradation mechanisms. Furthermore, MKRN1 depletion markedly curtailed the d-flow-induced decline in LATS1/2 and senescence, underscoring the involvement of the MKRN1 Ub E3 ligase in regulating LATS1/2 protein stability and d-flow-mediated EC senescence. CHX runoff assays following MKRN1 or control siRNA transfection revealed that MKRN1 is pivotal for LATS1/2 stability. The interaction between TERF2IP and MKRN1, enhanced by d-flow and attenuated by the TERF2IP S205A mutation, along with the inhibitory effect of TERF2IP c-myb domain overexpression on their association, indicates a binding site for MKRN1 on TERF2IP. Persistent TERF2IP-LATS1/2 binding until LATS1/2 expression declined suggests the formation of a TERF2IP-MKRN1-LATS1/2 complex that facilitates LATS1/2 degradation mediated by the recruitment of TERF2IP-MKRN1 complex. Conclusion: D-flow triggers the phosphorylation of TERF2IP at S205, leading to the formation of a TERF2IP-MKRN1-LATS1/2 complex. This complex initiates the degradation of LATS1/2, a key regulator of EC senescence. Thus, TERF2IP S205 phosphorylation under d-flow conditions is a crucial step in the pathway leading to LATS1/2 proteolysis, impacting our understanding of EC senescence and atherothrombosis.