Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers.

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the "simultaneous inhibition of a paralog pair" concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.

Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

Involvement of cochlin binding to sulfated heparan sulfate/heparin in the pathophysiology of autosomal dominant late-onset hearing loss (DFNA9).

Late-onset non-syndromic autosomal dominant hearing loss 9 (DFNA9) is a hearing impairment caused by mutations in the coagulation factor C homology gene (COCH). COCH encodes for cochlin, a major component of the cochlear extracellular matrix. Though biochemical and genetic studies have characterized the properties of wild-type and mutated cochlins derived from DFNA9, little is known about the underlying pathogenic mechanism. In this study, we established a cochlin reporter cell, which allowed us to monitor the interaction of cochlin with its ligand(s) by means of a ß-galactosidase assay. We found a class of highly sulfated glycosaminoglycans (GAGs), heparin, that were selectively bound to cochlin. The interaction was distinctly abrogated by N-desulfation, but not by 2-O- or 6-O-desulfation. The binding of cochlin to GAG was diminished by all of the point mutations found in DFNA9 patients. Through GAG composition analysis and immunostaining using mouse cochlin/immunoglobulin-Fc fusion protein, we identified moderately sulfated GAGs in mouse cochlea tissue; this implies that cochlin binds to such sulfated GAGs in the cochlea. Since GAGs play an important role in cell growth and survival as co-receptors of signal transduction mechanisms, the interaction of cochlin with GAGs in the extracellular matrix could aid the pathological research of autosomal dominant late-onset hearing loss in DFNA9.

Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation.

SignificanceThe nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that forms an inflammasome. The cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950 has established the structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation and the mechanism of action of the NLRP3 specific inhibitor. The inactive NLRP3 oligomer represents the NLRP3 resting state, capable of binding to membranes and is likely disrupted for its activation. Visualization of the inhibitor binding mode will enable optimization of the activity of NLRP3 inflammasome inhibitor drugs.

The structure of NLRP9 reveals a unique C-terminal region with putative regulatory function.

Nucleotide-binding and oligomerisation domain-like receptors (NLRs) can form inflammasomes that activate caspase-1 and pro-interleukin-1ß and induce pyroptosis. NLR family pyrin domain-containing 9 (NLRP9) forms an inflammasome and activates innate immune responses during virus infection, but little is known about this process. Here, we report the crystal and cryo-electron microscopy structures of NLRP9 in an ADP-bound state, revealing inactive and closed conformations of NLRP9 and its similarities to other structurally characterised NLRs. Moreover, we found a C-terminal region interacting with the concave surface of the leucine-rich repeat domain of NLRP9. This region is unique among NLRs and might be involved in the specific function of NLRP9. These data provide the structural basis for understanding the mechanism of NLRP9 regulation and activation.

The crystal structure of human DEAH-box RNA helicase 15 reveals a domain organization of the mammalian DEAH/RHA family.

DEAH-box RNA helicase 15 (DHX15) plays important roles in RNA metabolism, including in splicing and in ribosome biogenesis. In addition, mammalian DHX15 also mediates the innate immune sensing of viral RNA. However, structural information on this protein is not available, although the structure of the fungal orthologue of this protein, Prp43, has been elucidated. Here, the crystal structure of the ADP-bound form of human DHX15 is reported at a resolution of 2.0 Å. This is the first structure to be revealed of a member of the mammalian DEAH-box RNA helicase (DEAH/RHA) family in a nearly complete form, including the catalytic core consisting of the two N-terminal RecA domains and the C-terminal regulatory domains (CTD). The ADP-bound form of DHX15 displayed a compact structure, in which the RecA domains made extensive contacts with the CTD. Notably, a potential RNA-binding site was found on the surface of a RecA domain with positive electrostatic potential. Almost all structural features were conserved between the fungal Prp43 and the human DHX15, suggesting that they share a fundamentally common mechanism of action and providing a better understanding of the specific mammalian functions of DHX15.

Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner.

Upstream open reading frames (uORFs) are often found in the 5'-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression.

Polyamine-responsive ribosomal arrest at the stop codon of an upstream open reading frame of the AdoMetDC1 gene triggers nonsense-mediated mRNA decay in Arabidopsis thaliana.

During mRNA translation, nascent peptides with certain specific sequences cause arrest of ribosomes that have synthesized themselves. In some cases, such ribosomal arrest is coupled with mRNA decay. In yeast, mRNA quality control systems have been shown to be involved in mRNA decay associated with ribosomal arrest. However, a link between ribosomal arrest and mRNA quality control systems has not been found in multicellular organisms. In this study, we aimed to explore the relationship between ribosomal arrest and mRNA decay in plants. For this purpose, we used an upstream open reading frame (uORF) of the Arabidopsis thaliana AdoMetDC1 gene, in which the uORF-encoded peptide is involved in polyamine-responsive translational repression of the main coding sequence. Our in vitro analyses revealed that the AdoMetDC1 uORF-encoded peptide caused ribosomal arrest at the uORF stop codon in response to polyamine. Using transgenic calli harboring an AdoMetDC1 uORF-containing reporter gene, we showed that polyamine promoted mRNA decay in a uORF sequence-dependent manner. These results suggest that the polyamine-responsive ribosomal arrest mediated by the uORF-encoded peptide is coupled with mRNA decay. Our results also showed that the polyamine-responsive acceleration of mRNA decay was compromised by defects in factors that are essential for nonsense-mediated mRNA decay (NMD), an mRNA quality control system that degrades mRNAs with premature stop codons, suggesting that NMD is involved in AdoMetDC1 uORF peptide-mediated mRNA decay. Collectively, these findings suggest that AdoMetDC1 uORF peptide-mediated ribosomal arrest at the uORF stop codon induces NMD.