Summary Elucidation of endogenous cellular protein-protein interactions and their networks is most desirable for biological studies. Here we report our study of endogenous human coregulator protein complex networks obtained from integrative mass spectrometry-based analysis of 3,290 affinity purifications. By preserving weak protein interactions during complex isolation and utilizing high levels of reciprocity in the large dataset we identified many unreported protein associations, such as a transcriptional network formed by ZMYND8, ZNF687 and ZNF592. Furthermore, our work revealed a tiered interplay within networks that share common proteins, providing a conceptual organization of a cellular proteome composed of minimal endogenous modules (MEMOs), functional uniCOREs and regulatory complex-complex interaction networks (CCIs). This resource will effectively fill a void in linking correlative genomic studies with an understanding of transcriptional regulatory protein functions within the proteome for formulation and testing of new hypotheses.

Inactivating mutations in the thyroid hormone (TH) transporter Monocarboxylate transporter 8 (MCT8) cause severe psychomotor retardation in children. Animal models do not reflect the biology of the human disease. Using patient-specific induced pluripotent stem cells (iPSCs), we generated MCT8-deficient neural cells that showed normal TH-dependent neuronal properties and maturation. However, the blood-brain barrier (BBB) controls TH entry into the brain, and reduced TH availability to neural cells could instead underlie the diseased phenotype. To test potential BBB involvement, we generated an iPSC-based BBB model of MCT8 deficiency, and we found that MCT8 was necessary for polarized influx of the active form of TH across the BBB. We also found that a candidate drug did not appreciably cross the mutant BBB. Our results therefore clarify the underlying physiological basis of this disorder, and they suggest that circumventing the diseased BBB to deliver active TH to the brain could be a viable therapeutic strategy.

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In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is upregulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G 1 cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.T he p53 tumor suppressor is a key regulator of cell cycle arrest and apoptosis in response to genotoxic stress (1-3). The G 1 cell cycle checkpoint is operated through the p53-dependent transcriptional response (4). Accumulation of the p53 target gene product p21 WAF1∕CIP1 after DNA damage to a suprathreshold level, capable of blocking the G 1 -S promoting cyclinE/Cdk2 activity, may require several hours and is responsible for the sustained G 1 arrest (5).p53 is primarily regulated at the level of protein stability (6, 7). At least five E3 ligases (E6-AP, Mdm2, Arf-BP1, COP1, and Pirh2) have been identified to mediate ubiquitin-dependent proteasomal degradation of p53. Each of the E3 ligases is capable of building K48-linked polyubiquitin chains on p53, which are recognized by 26S proteasome for degradation. In response to DNA damage, it is thought that such polyubiquitination is inhibited to stabilize p53 protein. Mdm2 is the major p53 E3 ligase, because embryonic lethality of Mdm2-knockout mice caused by p53-induced apoptosis is rescued by deletion of p53 (8, 9).One paradox exists in the Mdm2 axis for p53 stabilization. Because Mdm2 is a p53 transcription target, stabilization of p53 leads to up-regulation of Mdm2 (10), which in turn should degrade p53, but p53 is maintained at high levels when the G 1 checkpoint is active. It was proposed that DNA damage destabilizes Mdm2 by a mechanism involving phosphorylation by ATM/ ATR and increased Mdm2 turnover. Thus, accelerated Mdm2 ubiquitination shortens its half-life, suppressing its activity towards p53 (11). Posttranslational modification of p53 is another important mechanism for its regulation (6). p53 modification by phosphorylation, acetylation, ...