Restoring the tumor suppressive activity of the Hippo signaling pathway lost through dysregulation of the NUAK1/2 and MARK2/3 kinase axis and downstream transcriptional effectors YAP/TAZ has emerged as a new modality for the treatment of several human cancers. Small molecule inhibition of NUAK1/2 and MARK2/3 constitutes a rational approach to block YAP/TAZ nuclear translocation and prevent a pro-oncogenic gene expression program. Modest structural changes to lead compound OICR14489, discovered through computational studies using a NUAK2 homology model, afforded the potent and selective dual NUAK1/2 and MARK2/3 inhibitor OICR16422. Further optimization led to inhibitor OICR19451, which produced an increase in YAP phosphorylation and enhanced cytoplasmic YAP/TAZ localization. In vitro growth inhibition of several cancer cell lines, coupled with the robust in vivo pharmacokinetic properties of OICR19451 marked it as an advanced tool compound suitable for in vivo evaluation. Accordingly, in an orthotopic model of highly metastatic breast cancer, MDA-MB-231 tumor-bearing mice treated with OICR19451 showed reduced metastases, tumor encapsulation and an overall increase in survival indicative of favorable Hippo pathway modulation.

Highly mutable pathogens generate viral diversity that impacts virulence, transmissibility, treatment, and thwarts acquired immunity. We previously described C19-SPAR-Seq, a high-throughput, next-generation sequencing platform to detect SARS-CoV-2 that we here deployed to systematically profile variant dynamics of SARS-CoV-2 for over 3 years in a large, North American urban environment (Toronto, Canada). Sequencing of the ACE2 receptor binding motif and polybasic furin cleavage site of the Spike gene in over 70,000 patients revealed that population sweeps of canonical variants of concern (VOCs) occurred in repeating wavelets. Furthermore, we found that VOC mutant derivatives and putative quasispecies that targeted functionally important residues and were found in future VOCs arose frequently, but were always extinguished. Systematic screening of functionally relevant domains in pathogens could thus provide a powerful tool for monitoring spread and mutational trajectories, particularly those with zoonotic potential.

Significance The OAS–RNase L system is an innate immunity pathway activated by viral infection. Viral dsRNA stimulates OAS enzymes to produce short 2′,5′-oligoadenylates (2-5A) that activate RNase L, resulting in cleavage of single-stranded (ss) RNA. We discovered a small-molecule inhibitor of RNase L that rescues the toxic phenotype of cells deficient in the dsRNA-editing enzyme ADAR1. ADAR1 destabilizes dsRNA to prevent OAS activity. ADAR1 mutations are responsible for a subset of cases of Aicardi-Goutières syndrome (AGS), a severe neurodevelopmental and inflammatory genetic disease of children with no effective medical therapy. We posit that an RNase L inhibitor may have utility against cases of AGS in which RNase L is activated and other indications where overactivation of RNase L is harmful.

Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.

Cilia are hair-like cellular protrusions important in many aspects of eukaryotic biology. For instance, motile cilia enable fluid movement over epithelial surfaces, while primary (sensory) cilia play roles in cellular signalling. The molecular events underlying cilia dynamics, and particularly their disassembly, are not well understood. Phosphatase and tensin homologue (PTEN) is an extensively studied tumour suppressor, thought to primarily act by antagonizing PI3-kinase signalling. Here we demonstrate that PTEN plays an important role in multicilia formation and cilia disassembly by controlling the phosphorylation of Dishevelled (DVL), another ciliogenesis regulator. DVL is a central component of WNT signalling that plays a role during convergent extension movements, which we show here are also regulated by PTEN. Our studies identify a novel protein substrate for PTEN that couples PTEN to regulation of cilia dynamics and WNT signalling, thus advancing our understanding of potential underlying molecular etiologies of PTEN-related pathologies.

A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the transforming growth factor β (TGF-β) pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex composed of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGF-β pathway (SMAD2/3), and the pluripotency regulator OCT4 (TSO). TSO collaborates with NuRD repressor complexes to buffer pluripotency gene expression while suppressing ME genes. Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements, and upon fate specification we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch-enhancer elements and provide a framework to understand how cellular context dictates interpretation of the same morphogen signal in development.

Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC50 (EC50=1μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway.

Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT domain.

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin ( Ub) protein ligases ( E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta ( TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.

The Rho family of small GTPases plays a key role in the dynamic regulation of the actin cytoskeleton that underlies various important cellular functions such as shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, could specifically target RhoA but not Cdc42 or Rac1 for degradation. Smurf1 interacts with the dominant inactive form of RhoA, RhoA N19, which binds constitutively to guanine nucleotide exchange factors (GEFs) in vivo. Smurf1 also interacts directly with either nucleotide-free or GDP-bound RhoA in vitro; however, loading with GTPgammaS inhibits the interaction. RhoA is ubiquitinated by wild-type Smurf1 but not the catalytic mutant of Smurf1 (C699A) in vivo and in vitro, indicating that RhoA is a direct substrate of Smurf1. In this chapter, we summarize the systems and methods used in the analyses of Smurf1-regulated RhoA ubiquitination and degradation.

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

2432 The small Rho family GTPases are key regulators of cytoskeletal dynamics that control cell shape, polarity and motility. Recent studies show that PAR6, which binds to atypical protein kinase C zeta (PKCζ), is key effector of Cdc42 and Rac1 and important for cell transformation, cell polarity, and regulation of epithelial tight junctions. However, it is still not clear how these biological functions are controlled by the Cdc42/Rac1-PAR6-PKCζ. The Smurf C2-WW-HECT domain ubiquitin ligases were recently shown to regulate TGFbeta signal transduction by targeting Smad signaling molecules for degradation. Here we demonstrated that Smurf1, a C2-WW-HECT domain E3 ubiquitin ligase, regulated cell polarity and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Overexpression of Smurf1 in Mv1Lu cells induced large number of dynamic protrusions, while moderate levels of expression of Smurf1 in NIH3T3 cells disrupted cell polarity. We also found that Smurf1 interacted with PKCζ, an effector of the Cdc42/Rac1-PAR6 polarity complex, and colocalized at cellular protrusions. The localization of Smurf1/ PKCζ complex was dependent on the PKCζ activity, while the expression of Smurf1 was also crucial for PKCζ localization at the protrusions. Furthermore, we showed that Smurf1 targeted RhoA for degradation through ubiquitin dependent proteasome pathway and therefore controlled the local level of RhoA, which is important for the local actin cytoskeleton dynamics. Thus, Smurf1 may link the polarity complex to degradation of RhoA in lamellipodia and filopodia to prevent RhoA signalling during dynamic membrane movements.

The Rho family of small guanosine triphosphatases regulates actin cytoskeleton dynamics that underlie cellular functions such as cell shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, regulated cell polarity and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Atypical protein kinase C zeta (PKCζ), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited Smurf1 to cellular protrusions, where it controlled the local level of RhoA. Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia and filopodia to prevent RhoA signaling during dynamic membrane movements.

Malnutrition remains a major problem of the developing countries in the world. In a programme aimed at controlling malnutrition in Nigeria, a newly formulated cereal-based food called 'ogi' with improved nutritional qualities has been developed using Lactobacillus acidophilus (DK 52) and Lactobacillus pentosus (DK 77) as starter cultures. The reducing sugar, protein content and essential amino acids were significantly higher in the newly developed 'ogi' than in the locally prepared one. The new ogi is thus more nutritious and has the potential to reduce malnourishment in our environment.

The immuno-polymerase chain reaction (PCR) approaches facilitate rapid (8 h) detection of Escherichia coli O157:H7 in contaminated dairy products and ground beef samples with detection sensitivities approaching 1 colony forming unit (cfu) g−1 ml−1. However, no PCR products were obtained when the method was applied to identify E. coli O157:H7 in tainted apple juice. Enzyme-linked immuno-assay (ELISA) results suggested non-specific binding of endogenous polyphenols (ubiquitous in plant products) to antibodies present on the surface of the immunobeads, making the latter unavailable for capturing the target bacteria. Treatment of the test sample, prior to IMS, with a synthetic fining agent, polyvinylpyrrolidone, restored the full function and sensitivity of the immuno-PCR. The study demonstrates the suitability of the improved method as a generic strategy for rapid screening of fruit juices and plant produce for E. coli O157:H7.