SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.
Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCFFbw7 and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation.
Objective: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL ), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. Conclusions: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.
Arterial Calcification due to Deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5′-nucleotidase (cluster of differentiation 73, CD73) enzyme. ACDC patients develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that ACDC patient vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor β (TGFβ) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFβ regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFβ signaling in vascular smooth muscle cells (SMCs). We show that Nt5e−/− SMCs have elevated contractile markers and elastin gene expression compared to Nt5e+/+ SMCs. Nt5e-deficient SMCs exhibit increased TGFβ-2 and activation of SMAD signaling, elevated elastin transcript and protein, and potentiates SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFβ signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFβ signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.
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