Summary
The Twist1 transcription factor is known to promote tumor metastasis and induce Epithelial-Mesenchymal Transition (EMT). Here, we report that Twist1 is capable of promoting the formation of invadopodia, specialized membrane protrusions for extracellular matrix degradation. Twist1 induces PDGFRα expression, which in turn activates Src, to promote invadopodia formation. We show that Twist1 and PDGFRα are central mediators of invadopodia formation in response to various EMT-inducing signals. Induction of PDGFRα and invadopodia is essential for Twist1 to promote tumor metastasis. Consistent with PDGFRα being a direct transcriptional target of Twist1, coexpression of Twist1 and PDGFRα predicts poor survival in breast tumor patients. Therefore, invadopodia-mediated matrix degradation is a key function of Twist1 in promoting tumor metastasis.
Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides.
Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI) - an important target for the development of new anti-staphylococcal drugs - as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme.
SUMMARYThe bones of the mammalian skull vault form through intramembranous ossification. Skull bones ossify directly, in a process regulated by -catenin, instead of passing through a cartilage intermediate. We tested whether -catenin is necessary for fate selection of intramembranous bone progenitors in the skull. Here, we show in mice that removal of -catenin from skull bone progenitors results in the near complete transformation of the skull bones to cartilage, whereas constitutive -catenin activation inhibits skull bone fate selection. -catenin directly activated Twist1 expression in skull progenitors, conditional Twist1 deletion partially phenocopied the absence of -catenin, and Twist1 deletion partially restored bone formation in the presence of constitutive -catenin activation. Finally, Twist1 bound robustly to the 3ЈUTR of Sox9, the central initiator of chondrogenesis, suggesting that Twist1 might directly repress cartilage formation through Sox9. These findings provide insight into how -catenin signaling via Twist1 actively suppresses the formation of cartilage and promotes intramembranous ossification in the skull.
Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16–19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.
Structured non-coding RNAs underline fundamental cellular processes, but determining their 3D structures remains challenging. We demonstrate herein that integrating NMR 1H chemical shift data with Rosetta de novo modeling can consistently return high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 blind targets, Chemical-Shift-ROSETTA for RNA (CS-ROSETTA-RNA) recovered the experimental structures with high accuracy (0.6 to 2.0 Å all-heavy-atom rmsd) in 18 cases.
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