Multiphase coalescence mediates Hippo pathway activation.

The function of biomolecular condensates is often restricted by condensate dissolution. Whether condensates can be suppressed without condensate dissolution is unclear. Here, we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and their coalescence into a common phase provides a mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inhibit STRIPAK function. These findings provide a paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a conceptual framework for understanding upstream regulation of the Hippo signaling pathway.

Calpain Cleaves Most Components in the Multiple Aminoacyl-tRNA Synthetase Complex and Affects Their Functions.

Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNA(Gln) and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca(2+) and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC.

Identification of lethal mutations in yeast threonyl-tRNA synthetase revealing critical residues in its human homolog.

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs.

A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity.

Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNA(Leu). LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain-domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS.

Polymorphism of stromal cell-derived factor-1 selectively upregulates gene expression and is associated with increased susceptibility to coronary artery disease.

Stromal cell-derived factor-1 (SDF-1) plays critical roles in vascular development and hematopoiesis. Here, we investigated the function of SDF-1 rs1801157G/A polymorphism in various immune cells and examined its association with susceptibility to coronary artery disease (CAD). Protein and mRNA levels of SDF-1 were tested in peripheral CD4+ T cell, CD8+ T cells, monocytes, and natural killer (NK) T cells from healthy donors with different genotypes of rs1801157G/A polymorphism. Prevalence of the polymorphism was compared between CAD patients and healthy controls. Data revealed that SDF-1 mRNA and protein were detectable in CD4+ T cells, CD8+ T cells, monocytes and NK T cells. Interestingly, both protein level and mRNA level of SDF-1 were significantly increased in the monocytes with rs1801157AA genotype, whereas the same phenomenon was not observed in the other three cell types. Blockage of CD14 completely inhibited the upregulation of SDF-1 in the monocytes with rs1801157AA genotype. Association analysis showed that frequencies of the rs1801157AA genotype and A allele were significantly higher in CAD cases than in controls (odds ratio [OR]=2.28, 95% confidence interval [CI], 1.50-3.29, p<0.0001, and OR=1.46, 95% CI, 1.21-3.73, p<0.0001, respectively). Also, prevalence of rs1801157AA genotype was further increased in cases with ST-elevation myocardial infarction (OR=1.65, 95% CI, 1.04-2.56, p=0.028). Our data suggest a novel pathway for regulating SDF-1 and a new risk factor for CAD.

Role of tRNA amino acid-accepting end in aminoacylation and its quality control.

Aminoacyl-tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-tRNAs. Editing occurs after translocation of the aminoacyl-CCA76 end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA76 accepting end of tRNA(Leu) during the aminoacylation and editing reactions. The analysis showed that the terminal A76 is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C74 and C75 selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA76 binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA76 homogeneity of tRNAs.

Hemin binds to human cytoplasmic arginyl-tRNA synthetase and inhibits its catalytic activity.

The free form of human cytoplasmic arginyl-tRNA synthetase (hcArgRS) is hypothesized to participate in ubiquitin-dependent protein degradation by offering arginyl-tRNA(Arg) to arginyl-tRNA transferase (ATE1). We investigated the effect of hemin on hcArgRS based on the fact that hemin regulates several critical proteins in the "N-end rule" protein degradation pathway. Extensive biochemical evidence has established that hemin could bind to both forms of hcArgRS in vitro. Based on the spectral changes of the Soret band on site-directed protein mutants, we identified Cys-115 as a specific axial ligand of hemin binding that is located in the Add1 domain. Hemin inhibited the catalytic activity of full-length and N-terminal 72-amino acid-truncated hcArgRSs by blocking amino acid activation. Kinetic analysis demonstrated that the K(m) values for tRNA(Arg), arginine, and ATP in the presence of hemin were not altered, but k(cat) values dramatically decreased compared with those in the absence of hemin. By comparison, the activity of prokaryotic ArgRS was not affected obviously by hemin. Gel filtration chromatography suggested that hemin induced oligomerization of both the isolated Add1 domain and the wild type enzyme, which could account for the inhibition of catalytic activity. However, the catalytic activity of an hcArgRS mutant with Cys-115 replaced by alanine (hcArgRS-C115A) was also inhibited by hemin, suggesting that hemin binding to Cys-115 is not responsible for the inhibition of enzymatic activity and that the specific binding may participate in other biological functions.

Spectrin couples cell shape, cortical tension, and Hippo signaling in retinal epithelial morphogenesis.

Although extracellular force has a profound effect on cell shape, cytoskeleton tension, and cell proliferation through the Hippo signaling effector Yki/YAP/TAZ, how intracellular force regulates these processes remains poorly understood. Here, we report an essential role for spectrin in specifying cell shape by transmitting intracellular actomyosin force to cell membrane. While activation of myosin II in Drosophila melanogaster pupal retina leads to increased cortical tension, apical constriction, and Yki-mediated hyperplasia, spectrin mutant cells, despite showing myosin II activation and Yki-mediated hyperplasia, paradoxically display decreased cortical tension and expanded apical area. Mechanistically, we show that spectrin is required for tethering cortical F-actin to cell membrane domains outside the adherens junctions (AJs). Thus, in the absence of spectrin, the weakened attachment of cortical F-actin to plasma membrane results in a failure to transmit actomyosin force to cell membrane, causing an expansion of apical surfaces. These results uncover an essential mechanism that couples cell shape, cortical tension, and Hippo signaling and highlight the importance of non-AJ membrane domains in dictating cell shape in tissue morphogenesis.

Nerfin-1 represses transcriptional output of Hippo signaling in cell competition.

The Hippo tumor suppressor pathway regulates tissue growth in Drosophila by restricting the activity of the transcriptional coactivator Yorkie (Yki), which normally complexes with the TEF/TEAD family DNA-binding transcription factor Scalloped (Sd) to drive the expression of growth-promoting genes. Given its pivotal role as a central hub in mediating the transcriptional output of Hippo signaling, there is great interest in understanding the molecular regulation of the Sd-Yki complex. In this study, we identify Nerfin-1 as a transcriptional repressor that antagonizes the activity of the Sd-Yki complex by binding to the TEA DNA-binding domain of Sd. Consistent with its biochemical function, ectopic expression of Nerfin-1 results in tissue undergrowth in an Sd-dependent manner. Conversely, loss of Nerfin-1 enhances the ability of winner cells to eliminate loser cells in multiple scenarios of cell competition. We further show that INSM1, the mammalian ortholog of Nerfin-1, plays a conserved role in repressing the activity of the TEAD-YAP complex. These findings reveal a novel regulatory mode converging on the transcriptional output of the Hippo pathway that may be exploited for modulating the YAP oncoprotein in cancer and regenerative medicine.

Homeostatic Control of Hpo/MST Kinase Activity through Autophosphorylation-Dependent Recruitment of the STRIPAK PP2A Phosphatase Complex.

The Hippo pathway controls organ size and tissue homeostasis through a kinase cascade leading from the Ste20-like kinase Hpo (MST1/2 in mammals) to the transcriptional coactivator Yki (YAP/TAZ in mammals). Whereas previous studies have uncovered positive and negative regulators of Hpo/MST, how they are integrated to maintain signaling homeostasis remains poorly understood. Here, we identify a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST. Mutation of the phospho-dependent docking sites in Hpo/MST or deletion of Slmap, the STRIPAK subunit recognizing these docking sites, results in constitutive activation of Hpo/MST in both Drosophila and mammalian cells. In contrast, autophosphorylation of the Hpo/MST linker at distinct sites is known to recruit Mats/MOB1 to facilitate Hippo signaling. Thus, multisite autophosphorylation of Hpo/MST linker provides an evolutionarily conserved built-in molecular platform to maintain signaling homeostasis by coupling antagonistic signaling activities.