Background: Metabolic syndrome is related to cardiovascular diseases, which is attributed in part, to arterial stiffness; however, the mechanisms remain unclear. The present study aimed to investigate the molecular mechanisms of metabolic syndrome–induced arterial stiffness and to identify new therapeutic targets. Methods: Arterial stiffness was induced by high-fat/high-sucrose diet in mice, which was quantified by Doppler ultrasound. Four-dimensional label-free quantitative proteomic analysis, affinity purification and mass spectrometry, and immunoprecipitation and GST pull-down experiments were performed to explore the mechanism of YAP (Yes-associated protein)-mediated TGF (transforming growth factor) β pathway activation. Results: YAP protein was upregulated in the aortic tunica media of mice fed a high-fat/high-sucrose diet for 2 weeks and precedes arterial stiffness. Smooth muscle cell–specific YAP knockdown attenuated high-fat/high-sucrose diet–induced arterial stiffness and activation of TGFβ-Smad2/3 signaling pathway in arteries. By contrast, Myh11Cre ERT2 -Yap Tg mice exhibited exacerbated high-fat/high-sucrose diet–induced arterial stiffness and enhanced TGFβ-activated Smad2/3 phosphorylation in arteries. PPM1B (protein phosphatase, Mg 2+ /Mn 2+ -dependent 1B) was identified as a YAP-bound phosphatase that translocates into the nucleus to dephosphorylate Smads in response to TGFβ. This process was inhibited by YAP through removal of the K63-linked ubiquitin chain of PPM1B at K326. Conclusions: This study provides a new mechanism by which smooth muscle cell YAP regulates the TGFβ pathway and a potential therapeutic target in metabolic syndrome–associated arterial stiffness.

Rationale: A number of guanine nucleotide exchange factors (GEFs) including epithelial cell transforming factor ECT2 are believed to drive carcinogenesis through activating distinct oncogenic GTPases. Yet, whether GEF-independent activity of ECT2 also plays a role in tumorigenesis remains unclear. Methods: Immunohistochemical (IHC) staining, colony formation and xenograft assays were used to examine the role of ECT2 in breast carcinogenesis. Co-immunoprecipitation, immunofluorescent stainings, in vivo deubiquitination and in vitro deubiquitination experiments were performed to examine the physical and functional interaction between ECT2 and ubiquitin-specific protease USP7. High-throughput RNA sequencing, quantitative reverse transcription-PCR and Western blotting were employed to investigate the biological significance of the interplay between ECT2 and USP7. Results: We report that ECT2 plays a tumor-promoting role in breast cancer, and GEF activity-deficient ECT2 is able to alleviate ECT2 depletion associated growth defects in breast cancer cells. Mechanistically, we demonstrated that ECT2 physically interacts with ubiquitin-specific protease USP7 and functionally facilitates USP7 intermolecular self-association, -deubiquitination and -stabilization in a GEF activity-independent manner. USP7 in turn, deubiquitinates and stabilizes ECT2, resulting in a feedforward regulatory circuit that ultimately sustains the expression of oncogenic protein MDM2. Conclusion: Our study uncovers a GEF-independent role of ECT2 in promoting survival of breast cancer cells, provides a molecular insight for the reciprocal regulation of ECT2 and USP7, and supports the pursuit of ECT2/USP7 as potential targets for breast cancer intervention.

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Background Single-stranded DNA (ssDNA) coated with replication protein A (RPA) acts as a key platform for the recruitment and exchange of genome maintenance factors in DNA damage response. Yet, how the formation of the ssDNA-RPA intermediate is regulated remains elusive. Results Here, we report that the lamin-associated protein LAP2α is physically associated with RPA, and LAP2α preferentially facilitates RPA deposition on damaged chromatin via physical contacts between LAP2α and RPA1. Importantly, LAP2α-promoted RPA binding to ssDNA plays a critical role in protection of replication forks, activation of ATR, and repair of damaged DNA. We further demonstrate that the preference of LAP2α-promoted RPA loading on damaged chromatin depends on poly ADP-ribose polymerase PARP1, but not poly(ADP-ribosyl)ation. Conclusions Our study provides mechanistic insight into RPA deposition in response to DNA damage and reveals a genome protection role of LAP2α.