Wenjia Wang

A Peptide Mimicking VGLL4 Function Acts as a YAP Antagonist Therapy against Gastric Cancer

The Hippo pathway has been implicated in suppressing tissue overgrowth and tumor formation by restricting the oncogenic activity of YAP. However, transcriptional regulators that inhibit YAP activity have not been well studied. Here, we uncover clinical importance for VGLL4 in gastric cancer suppression and find that VGLL4 directly competes with YAP for binding TEADs. Importantly, VGLL4s tandem Tondu domains are not only essential but also sufficient for its inhibitory activity toward YAP. A peptide mimicking this function of VGLL4 potently suppressed tumor growth in vitro and in vivo. These findings suggest that disruption of YAP-TEADs interaction by a VGLL4-mimicking peptide may be a promising therapeutic strategy against YAP-driven human cancers.

The kinase MST4 limits inflammatory responses through direct phosphorylation of the adaptor TRAF6

Immune responses need to be tightly controlled to avoid excessive inflammation and prevent unwanted host damage. Here we report that germinal center kinase MST4 responded dynamically to bacterial infection and acted as a negative regulator of inflammation. We found that MST4 directly interacted with and phosphorylated the adaptor TRAF6 to prevent its oligomerization and autoubiquitination. Accordingly, MST4 did not inhibit lipopolysaccharide-induced cytokine production in Traf6−/− embryonic fibroblasts transfected to express a mutant form of TRAF6 that cannot be phosphorylated at positions 463 and 486 (with substitution of alanine for threonine at those positions). Upon developing septic shock, mice in which MST4 was knocked down showed exacerbated inflammation and reduced survival, whereas heterozygous deletion of Traf6 (Traf6+/−) alleviated such deleterious effects. Our findings reveal a mechanism by which TRAF6 is regulated and highlight a role for MST4 in limiting inflammatory responses.

Striatins Contain a Noncanonical Coiled Coil That Binds Protein Phosphatase 2A A Subunit to Form a 2:2 Heterotetrameric Core of Striatin-interacting Phosphatase and Kinase (STRIPAK) Complex

Background: Striatins are novel regulatory subunits of PP2A in the striatin-interacting phosphatase and kinase (STRIPAK) complex. Results: The striatin coiled coil forms a noncanonical dimer required for PP2A A subunit binding. Conclusion: The coiled coil of striatins bind PP2A A subunits to form a 2:2 heterotetrameric core of the STRIPAK complex. Significance: The current structural analysis provides insights into the assembly of the STRIPAK complex. The protein phosphatase 2A (PP2A) and kinases such as germinal center kinase III (GCKIII) can interact with striatins to form a supramolecular complex called striatin-interacting phosphatase and kinase (STRIPAK) complex. Despite the fact that the STRIPAK complex regulates multiple cellular events, it remains only partially understood how this complex itself is assembled and regulated for differential biological functions. Our recent work revealed the activation mechanism of GCKIIIs by MO25, as well as how GCKIIIs heterodimerize with CCM3, a molecular bridge between GCKIII and striatins. Here we dissect the structural features of the coiled coil domain of striatin 3, a novel type of PP2A regulatory subunit that functions as a scaffold for the assembly of the STRIPAK complex. We have determined the crystal structure of a selenomethionine-labeled striatin 3 coiled coil domain, which shows it to assume a parallel dimeric but asymmetric conformation containing a large bend. This result combined with a number of biophysical analyses provide evidence that the coiled coil domain of striatin 3 and the PP2A A subunit form a stable core complex with a 2:2 stoichiometry. Structure-based mutational studies reveal that homodimerization of striatin 3 is essential for its interaction with PP2A and therefore assembly of the STRIPAK complex. Wild-type striatin 3 but not the mutants defective in PP2A binding strongly suppresses apoptosis of Jurkat cells induced by the GCKIII kinase MST3, most likely through a mechanism in which striatin recruits PP2A to negatively regulate the activation of MST3. Collectively, our work provides structural insights into the organization of the STRIPAK complex and will facilitate further functional studies.

Structural and biochemical studies of RIG-I antiviral signaling

Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.

A non‐canonical role of the p97 complex in RIG‐I antiviral signaling

RIG‐I is a well‐studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4‐Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG‐I. The p97 complex is able to directly bind both non‐ubiquitinated RIG‐I and the E3 ligase RNF125, promoting K48‐linked ubiquitination of RIG‐I at residue K181. Viral infection significantly strengthens the interaction between RIG‐I and the p97 complex by a conformational change of RIG‐I that exposes the CARDs and through K63‐linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG‐I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.

Germinal center kinases in immune regulation

Germinal center kinases (GCKs) participate in a variety of signaling pathways needed to regulate cellular functions including apoptosis, cell proliferation, polarity and migration. Recent studies have shown that GCKs are participants in both adaptive and innate immune regulation. However, the differential activation and regulatory mechanisms of GCKs, as well as upstream and downstream signaling molecules, remain to be fully defined. It remains unresolved whether and how GCKs may cross-talk with existing signaling pathways. This review stresses the progresses in research of GCKs relevant to the immune system.

Structure of MST2 SARAH domain provides insights into its interaction with RAPL.

The STE20 kinases MST1 and MST2 are key players in mammalian Hippo pathway. The SARAH domains of MST1/2 act as a platform to mediate homodimerization and hetero-interaction with a range of adaptors including RASSFs and Salvador, which also possess SARAH domains. Here, we determined the crystal structure of human MST2 SARAH domain, which forms an antiparallel homodimeric coiled coil. Structural comparison indicates that SARAH domains of different proteins may utilize a shared dimerization module to form homodimer or heterodimer. Structure-guided mutational study identified specific interface residues critical for MST2 homodimerization. MST2 mutations disrupting its homodimerization also impaired its hetero-interaction with RAPL (also named RASSF5 and NORE1), which is mediated by their SARAH domains. Further biochemical and cellular assays indicated that SARAH domain-mediated homodimerization and hetero-interaction with RAPL are required for full activation of MST2 and therefore apoptotic functions in T cells.

DNA-binding mechanism of the Hippo pathway transcription factor TEAD4

TEA domain (TEAD) family transcription factors are key regulators in development, tissue homeostasis and cancer progression. TEAD4 acts as a critical downstream effector of the evolutionarily conserved Hippo signaling pathway. The well-studied oncogenic protein YAP forms a complex with TEAD4 to regulate gene transcription; so does the tumor suppressor VGLL4. Although it is known that TEAD proteins can bind promoter regions of target genes through the TEA domain, the specific and detailed mechanism of DNA recognition by the TEA domain remains partially understood. Here, we report the crystal structure of TEAD4 TEA domain in complex with a muscle-CAT DNA element. The structure revealed extensive interactions between the TEA domain and the DNA duplex involving both the major and minor grooves of DNA helix. The DNA recognition helix, α3 helix, determines the specificity of the TEA domain binding to DNA sequence. Structure-guided biochemical analysis identified two major binding sites on the interface of the TEA domain–DNA complex. Mutation of TEAD4 at either site substantially decreases its occupancy on the promoter region of target genes, and largely impaired YAP-induced TEAD4 transactivation and target gene transcription, leading to inhibition of growth and colony formation of gastric cancer cell HGC-27. Collectively, our work provides a structural basis for understanding the regulatory mechanism of TEAD-mediated gene transcription.

The Transitional Endoplasmic Reticulum ATPase p97 Regulates the Alternative Nuclear Factor NF-κB Signaling via Partial Degradation of the NF-κB Subunit p100.

Background: In the alternative NF-κB signaling pathway, p100 undergoes partial degradation to generate p52. Results: Depletion of the p97-Npl4-Ufd1 complex or enzymatic inhibition of p97 significantly decreases the processing of p100 into p52. Conclusion: The p97-Npl4-Ufd1 complex positively regulates the alternative NF-κB signaling pathway. Significance: This work suggests that p97 is a potential target for diseases with dysregulation of the alternative NF-κB pathway. Partial degradation of the p100 subunit to generate p52 subunit is a hallmark of the alternative NF-κB pathway, which has been implicated in cancer. Here, we uncovered a role of the p97-Npl4-Ufd1 complex in mediating p100-to-p52 processing and therefore positively regulating the alternative NF-κB pathway. We observed an elevation of p97 mRNA levels in lymphoma patients, which positively correlates with NFKB2 expression, a downstream target gene of the alternative NF-κB pathway. Moreover, NFKB2 mRNA levels were aberrantly down-regulated in patients with inclusion body myopathy associated with Pagets disease of the bone and frontotemporal dementia (IBMPFD), a disease caused by mutation of p97. Inactivation of p97 or depletion of the p97-Npl4-Ufd1 complex inhibits the processing of p100 into p52, decreasing transcription of the downstream target genes. Further analyses reveal that the p97-Npl4-Ufd1 complex interacts with F-box and WD repeats protein SCFβTrCP complex to regulate the partial degradation of p100, a process involving K48- and K11-linked ubiquitination. In line with this, in LPS-induced lung damage mice model, generation of p52 is significantly decreased in p97-KD mice compared with mock mice. Finally, abrogation of p97 ATPase activity by its specific inhibitor DBeQ, efficiently decreased proliferation of lymphoma cells. Collectively, our study revealed a regulatory role of the p97-Npl4-Ufd1 complex in regulating p100 partial degradation, highlighting the potential of p97 as a drug target for cancers with aberrant activation of the alternative NF-κB pathway.

Structural and Biochemical Insights into the Activation Mechanisms of Germinal Center Kinase OSR1

Background: OSR1 modulates ion homeostasis and cell volume in mammal. Results: The CCT domain and αAL helix of OSR1 act together to suppress its basal activity, while WNKs and MO25 unlock such autoinhibition for full activation. Conclusion: OSR1 activity is regulated by autoinhibition, WNKs, and MO25. Significance: This work provides insights into the regulatory mechanisms of OSR1 activation to facilitate functional study. The oxidative stress-responsive 1 (OSR1) kinase belongs to the mammalian STE20-like kinase family. OSR1 is activated by with no lysine [K] (WNKs) kinases, and then it phosphorylates cation-coupled Cl-cotransporters, regulating ion homeostasis and cell volume in mammalian cells. However, the specific mechanisms of OSR1 activation remains poorly defined, largely due to its extremely low basal activity. Here, we dissect in detail the regulatory mechanisms of OSR1 activation from the aspects of autoinhibition, upstream kinase WNK, and the newly identified master regulator mouse protein-25 (MO25). Based on our structural and biochemical studies, we propose a “double lock” model, accounting for the tight autoinhibition of OSR1, an effect that has to be removed by WNK before MO25 further activates OSR1. Particularly, the conserved C-terminal (CCT) domain and αAL helix act together to strongly suppress OSR1 basal activity. WNKs bind to the CCT and trigger its conformational rearrangement to release the kinase domain of OSR1, allowing for MO25 binding and full activation. Finally, the regulatory mechanisms of OSR1 activation were further corroborated by cellular studies of OSR1-regulated cell volume control through WNK-OSR1 signaling pathway. Collectively, these results provide insights into the OSR1 kinase activation to facilitate further functional study.