Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.

In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions—a phenomenon we name ER to Cytosol Signaling (ERCYS).

Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down–based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mass spectrometry. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

Twelve polyphenols from three distinct families (dihydroflavonols, flavan-3-ols, and flavanones) were studied as potential substrates of anthocyanidin synthase from Vitis vinifera (VvANS). Only flavan-3-ols of (2R,3S) configuration having either a catechol or gallol group on ring B are accepted as substrates. Only dihydroflavonols of (2R,3R) configuration are accepted as substrates, but a catechol or gallol group is not mandatory. Flavanones are not substrates of VvANS. HPLC and MS/MS analyses of the enzymatic products showed that the VvANS-catalyzed oxidative transformation of (+)-dihydroflavonols, such as dihydroquercetin, dihydrokaempferol and dihydromyricetin, leads only to the corresponding flavonols. Among the flavan-3-ols recognized as substrates, (+)-gallocatechin was only transformed into delphinidin by VvANS, whereas (+)-catechin was transformed into three products, including two major products that were an ascorbate–cyanidin adduct and a dimer of oxidized catechin, and a minor product that was cyanidin. Data from real-time MS monitoring of the enzymatic transformation of (+)-catechin suggest that its products are all derived from the initial C₃-hydroxylation intermediate, i.e., a 3,3-gem-diol, and their most likely formation mechanism is discussed.