Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aß1-42). We found two compounds that were selective for TTR and did not disrupt Aß-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein.

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Characterization of Non-amyloidogenic G101S Transthyretin.

Transthyretin (TTR) is a tetrameric beta-sheet-rich protein that is important in the plasma transport of thyroxine and retinol. Mutations in the TTR gene cause TTR tetramer protein to dissociate to monomer, which is the rate-limiting step in familial amyloid polyneuropathy. Amyloidogenicity of individual TTR variants depends on the types of mutation that induce significant changes in biophysical, biochemical and/or biological properties. G101S TTR variant was previously identified in a Japanese male without amyloidotic symptom, and was considered as a non-amyloidogenic TTR variant. However, little is known about G101S TTR. Here, we found slight but possibly important biophysical differences between wild-type (WT) and G101S TTR. G101S TTR had slower rate of tetramer dissociation and lower propensity for amyloid fibril formation, especially at mild low pH (4.2 and 4.5), and was likely to have strong hydrophobic interaction among TTR monomers, suggesting relatively higher stability of G101S TTR compared with WT TTR. Cycloheximide (CHX)-based assay in HEK293 cells revealed that intracellular G101S TTR expression level was lower, but extracellular expression was higher than WT TTR, implying enhanced secretion efficiency of G101S TTR protein compared with WT TTR. Moreover, we found that STT3B-dependent posttranslational N-glycosylation at N98 residue occurred in G101S TTR but not in other TTR variants, possibly due to amino acid alterations that increase N-glycosylation preference or accelerate rigid structure formation susceptible to N-glycosylation. Taken together, our study characterizes G101S TTR as a stable and N-glycosylable TTR, which may be linked to its non-amyloidogenic characteristic.

Structural stabilization of transthyretin by a new compound, 6-benzoyl-2-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione.

Familial amyloid polyneuropathy (FAP) is a genetic, adult-onset, neurodegenerative disorder caused by amyloid formation of transthyretin (TTR), a thyroxine-binding protein. Mutation in TTR causes a propensity of TTR tetramer to dissociate to monomer, which is the first step to amyloidosis. Thus, a drug that can stabilize the tetramer structure will have therapeutic benefit. Here, by virtual screening and biochemical assays, we identified small molecule 6-benzoyl-2-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (L6) that can prevent the dissociation of TTR to monomer. X-ray crystallography reveals that L6 binds to the T4 binding pocket of TTR. These findings show that L6 is a candidate TTR stabilizer.

Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events.

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native "wildtype" DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach. Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells. Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

Differential Kat3 Usage Orchestrates the Integration of Cellular Metabolism with Differentiation.

The integration of cellular status with metabolism is critically important and the coupling of energy production and cellular function is highly evolutionarily conserved. This has been demonstrated in stem cell biology, organismal, cellular and tissue differentiation and in immune cell biology. However, a molecular mechanism delineating how cells coordinate and couple metabolism with transcription as they navigate quiescence, growth, proliferation, differentiation and migration remains in its infancy. The extreme N-termini of the Kat3 coactivator family members, CBP and p300, by far the least homologous regions with only 66% identity, interact with members of the nuclear receptor family, interferon activated Stat1 and transcriptionally competent ß-catenin, a critical component of the Wnt signaling pathway. We now wish to report based on multiomic and functional investigations, utilizing p300 knockdown, N-terminal p300 edited and p300 S89A edited cell lines and p300 S89A knockin mice, that the N-termini of the Kat3 coactivators provide a highly evolutionarily conserved hub to integrate multiple signaling cascades to coordinate cellular metabolism with the regulation of cellular status and function.

p300 Serine 89: A Critical Signaling Integrator and Its Effects on Intestinal Homeostasis and Repair.

Differential usage of Kat3 coactivators, CBP and p300, by ß-catenin is a fundamental regulatory mechanism in stem cell maintenance and initiation of differentiation and repair. Based upon our earlier pharmacologic studies, p300 serine 89 (S89) is critical for controlling differential coactivator usage by ß-catenin via post-translational phosphorylation in stem/progenitor populations, and appears to be a target for a number of kinase cascades. To further investigate mechanisms of signal integration effected by this domain, we generated p300 S89A knock-in mice. We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly increase the risk of developing colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in intestine of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution.