lncRNA BREA2 promotes metastasis by disrupting the WWP2-mediated ubiquitination of Notch1.

Notch has been implicated in human cancers and is a putative therapeutic target. However, the regulation of Notch activation in the nucleus remains largely uncharacterized. Therefore, characterizing the detailed mechanisms governing Notch degradation will identify attractive strategies for treating Notch-activated cancers. Here, we report that the long noncoding RNA (lncRNA) BREA2 drives breast cancer metastasis by stabilizing the Notch1 intracellular domain (NICD1). Moreover, we reveal WW domain containing E3 ubiquitin protein ligase 2 (WWP2) as an E3 ligase for NICD1 at K1821 and a suppressor of breast cancer metastasis. Mechanistically, BREA2 impairs WWP2-NICD1 complex formation and in turn stabilizes NICD1, leading to Notch signaling activation and lung metastasis. BREA2 loss sensitizes breast cancer cells to inhibition of Notch signaling and suppresses the growth of breast cancer patient-derived xenograft tumors, highlighting its therapeutic potential in breast cancer. Taken together, these results reveal the lncRNA BREA2 as a putative regulator of Notch signaling and an oncogenic player driving breast cancer metastasis.

Bip-Yorkie interaction determines oncogenic and tumor-suppressive roles of Ire1/Xbp1s activation.

Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. ER proteostasis is essential to adapt to cell proliferation and regeneration in development and tumorigenesis, but mechanisms linking UPR, growth control, and cancer progression remain unclear. Here, we report that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner. Activation of Ire1/Xbp1s up-regulates their downstream target Bip, which sequesters Yorkie (Yki), a Hippo pathway transducer, in the cytoplasm to restrict Yki transcriptional output. This regulation provides an endogenous defensive mechanism in organ size control, intestinal homeostasis, and regeneration. Unexpectedly, Xbp1 ablation promotes tumor overgrowth but suppresses invasiveness in a Drosophila cancer model. Mechanistically, hyperactivated Ire1/Xbp1s signaling in turn induces JNK-dependent developmental and oncogenic cell migration and epithelial-mesenchymal transition (EMT) via repression of Yki. In humans, a negative correlation between XBP1 and YAP (Yki ortholog) target gene expression specifically exists in triple-negative breast cancers (TNBCs), and those with high XBP1 or HSPA5 (Bip ortholog) expression have better clinical outcomes. In human TNBC cell lines and xenograft models, ectopic XBP1s or HSPA5 expression alleviates tumor growth but aggravates cell migration and invasion. These findings uncover a conserved crosstalk between the Ire1/Xbp1s and Hippo signaling pathways under physiological settings, as well as a crucial role of Bip-Yki interaction in tumorigenesis that is shared from Drosophila to humans.

The self-association and activity regulation of LRSAM1 E3 ligase.

LRSAM1, a RING-type E3 ubiquitin ligase, is essential for regulating cargo sorting, signaling pathways, cell adhesion and anti-bacterial autophagy. It is important to elucidate the mechanism that underlies the regulation of LRSAM1 E3 ligase activity. Here, we reported that LRSAM1 exhibited self-association in vitro and in vivo. We found the self-association of LRSAM1 promotes intermolecular ubiquitination and proved a potential N-terminal ubiquitination. The E3 activity of LRSAM1 is amplified when the RING domain is present in tandem with its N-terminal domain(s). Furthermore, we found that the CC2-SAM domain had a strong inhibitory effect on the E3 activity of LRSAM1 in vitro and blocked ubiquitination of TSG101 in vivo; the tandem CC1 domain, but not the individual CC1 domain, could counteract this inhibition. Collectively, our data characterized the self-association of LRSAM1 and showed how its domains may contribute to its overall activity.

Expression in Escherichia coli, purification and characterization of LRSAM1, a LRR and RING domain E3 ubiquitin ligase.

LRSAM1 is a typical RING-finger E3 ubiquitin ligase that plays an important role in many processes. The expression and purification of LRSAM1 from Escherichiacoli had not yet been reported. Here, strategies to clone, express and purify recombinant LRSAM1 in E. coli cells were developed. LRSAM1 was expressed with high yield as inclusion bodies and successfully recovered in soluble form by subsequent denaturation and renaturation steps. Refolded LRSAM1 was directly purified through two steps of ammonium sulfate precipitation, resulting in a purity of up to 95% and a yield of about 6 mg/L bacterial culture. Purified recombinant LRSAM1 exhibited a pH-dependent E3 ligase activity. Its ligase activity was RING-finger domain-dependent, and its ubiquitination favors K6-, K27-, K29- and K48-linkages in cooperation with UbcH5-type E2 enzymes.

A spatially defined human Notch receptor interaction network reveals Notch intracellular storage and Ataxin-2-mediated fast recycling.

The Notch signaling pathway controls cell growth, differentiation, and fate decisions. Dysregulation of Notch signaling has been linked to various human diseases. Notch receptor resides in multiple cellular compartments, and its translocation plays a central role in pathway activation. However, the spatial regulation of Notch receptor functions remains largely elusive. Using TurboID-based proximity labeling followed by affinity purification and mass spectrometry, we establish a spatially defined human Notch receptor interaction network. Notch receptors interact with different proteins in distinct subcellular compartments to perform specific cellular functions. This spatially defined interaction network also reveals that a large fraction of NOTCH is stored at the endoplasmic reticulum (ER)-Golgi intermediate compartment and recruits Ataxin-2-dependent recycling machinery for rapid recycling, Notch signaling activation, and leukemogenesis. Our work provides insights into dynamic Notch receptor complexes with exquisite spatial resolution, which will help in elucidating the detailed regulation of Notch receptors and highlight potential therapeutic targets for Notch-related pathogenesis.

Protocol for establishing a protein-protein interaction network using tandem affinity purification followed by mass spectrometry in mammalian cells.

Identification of protein interactors is fundamental to understanding their functions. Here, we describe a modified protocol for tandem affinity purification coupled with mass spectrometry (TAP/MS), which includes two-step purification. We detail the S-, 2×FLAG-, and Streptavidin-Binding Peptide (SBP)- tandem tags (SFB-tag) system for protein purification. This protocol can be used to identify protein interactors and establish a high-confidence protein-protein interaction network based on computational models. This is particularly useful for identifying bona fide interacting proteins for subsequent functional studies. For complete details on the use and execution of this protocol, please refer to Bian et al. (2021).

FBXO42 facilitates Notch signaling activation and global chromatin relaxation by promoting K63-linked polyubiquitination of RBPJ.

Dysregulation of the Notch-RBPJ (recombination signal-binding protein of immunoglobulin kappa J region) signaling pathway has been found associated with various human diseases including cancers; however, precisely how this key signaling pathway is fine-tuned via its interactors and modifications is still largely unknown. In this study, using a proteomic approach, we identified F-box only protein 42 (FBXO42) as a previously unidentified RBPJ interactor. FBXO42 promotes RBPJ polyubiquitination on lysine-175 via lysine-63 linkage, which enhances the association of RBPJ with chromatin remodeling complexes and induces a global chromatin relaxation. Genetically depleting FBXO42 or pharmacologically targeting its E3 ligase activity attenuates the Notch signaling-related leukemia development in vivo. Together, our findings not only revealed FBXO42 as a critical regulator of the Notch pathway by modulating RBPJ-dependent global chromatin landscape changes but also provided insights into the therapeutic intervention of the Notch pathway for leukemia treatment.

AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells.

The current coronavirus disease 2019 (COVID-19) pandemic presents a global public health challenge. The viral pathogen responsible, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the host receptor ACE2 through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. Although the role of ACE2 as a receptor for SARS-CoV-2 is clear, studies have shown that ACE2 expression is extremely low in various human tissues, especially in the respiratory tract. Thus, other host receptors and/or co-receptors that promote the entry of SARS-CoV-2 into cells of the respiratory system may exist. In this study, we found that the tyrosine-protein kinase receptor UFO (AXL) specifically interacts with the N-terminal domain of SARS-CoV-2 S. Using both a SARS-CoV-2 virus pseudotype and authentic SARS-CoV-2, we found that overexpression of AXL in HEK293T cells promotes SARS-CoV-2 entry as efficiently as overexpression of ACE2, while knocking out AXL significantly reduces SARS-CoV-2 infection in H1299 pulmonary cells and in human primary lung epithelial cells. Soluble human recombinant AXL blocks SARS-CoV-2 infection in cells expressing high levels of AXL. The AXL expression level is well correlated with SARS-CoV-2 S level in bronchoalveolar lavage fluid cells from COVID-19 patients. Taken together, our findings suggest that AXL is a novel candidate receptor for SARS-CoV-2 which may play an important role in promoting viral infection of the human respiratory system and indicate that it is a potential target for future clinical intervention strategies.

Low-density-lipoprotein-receptor-related protein 1 mediates Notch pathway activation.

The Notch signaling pathway controls cell growth, differentiation, and fate decisions, and its dysregulation has been linked to various human genetic disorders and cancers. To comprehensively understand the global organization of the Notch pathway and identify potential drug targets for Notch-related diseases, we established a protein interaction landscape for the human Notch pathway. By combining and analyzing genetic and phenotypic data with bioinformatics analysis, we greatly expanded this pathway and identified many key regulators, including low-density-lipoprotein-receptor-related protein 1 (LRP1). We demonstrated that LRP1 mediates the ubiquitination chain linkage switching of Delta ligands, which further affects ligand recycling, membrane localization, and stability. LRP1 inhibition led to Notch signaling inhibition and decreased tumorigenesis in leukemia models. Our study provides a glimpse into the Notch pathway interaction network and uncovers LRP1 as one critical regulator of the Notch pathway, as well as a possible therapeutic target for Notch-related cancers.

Michael addition of dehydroalanine-containing MAPK peptides to catalytic lysine inhibits the activity of phosphothreonine lyase.

The phosphothreonine lyases OspF and SpvC irreversibly inactivate host dual-phosphorylated mitogen-activated protein kinases (MAPKs) [pThr-X-pTyr motif] through ß-elimination. We found that dual-phosphorylated (pSer-X-pTyr) MAPK substrate peptides and their resulting catalytic products cross-link to OspF and SpvC. Mass spectrometry results revealed that these linkages form between lysine, which acts as a general base, and dehydroalanine (Dha) on catalytic products. The nucleophilic addition efficiency is dependent on the K136 residue being in a deprotonated state. Peptide cross-linking inhibits the activity of SpvC and blocks the inactivation of MAPK signaling by SpvC. Small compounds mimicking these sequences may act as phosphothreonine lyase inhibitors.