Summary An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved with Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into the embryonic gut tube and can form pancreatic progenitors in vitro. The application of small molecules to differentiate mouse and human ESCs into endoderm, and pancreatic progenitors represents a step toward achieving a reproducible and efficient production of desired ES cell derivatives.

To study the genetic control of plant responses to cold stress, Arabidopsis thaliana mutants were isolated by a screen for mutations that impair cold-induced transcription of the CBF3-LUC reporter gene. We report here the characterization and cloning of a mutated gene, atnup160-1, which causes reduced CBF3-LUC induction under cold stress. atnup160-1 mutant plants display altered cold-responsive gene expression and are sensitive to chilling stress and defective in acquired freezing tolerance. AtNUP160 was isolated through positional cloning and shown to encode a putative homolog of the animal nucleoporin Nup160. In addition to the impaired expression of CBF genes, microarray analysis revealed that a number of other genes important for plant cold tolerance were also affected in the mutants. The atnup160 mutants flower early and show retarded seedling growth, especially at low temperatures. AtNUP160 protein is localized at the nuclear rim, and poly(A)-mRNA in situ hybridization shows that mRNA export is defective in the atnup160-1 mutant plants. Our study suggests that Arabidopsis AtNUP160 is critical for the nucleocytoplasmic transport of mRNAs and that it plays important roles in plant growth and flowering time regulation and is required for cold stress tolerance.In eukaryotic cells, the genome is enclosed within the nucleus. Nucleocytoplasmic transport of macromolecules across the nuclear membrane occurs through channels formed by nuclear pore complexes (NPCs). Embedded in the double-lipid bilayer nuclear envelope, NPCs form a ringlike structure surrounding a central pore that is believed to facilitate the bidirectional transport of RNAs, proteins, and ribonucleoprotein particles and, at the same time, to allow the diffusion of small molecules and ions across the double membrane (reviewed in reference 4). The overall three-dimensional architecture and transport mechanisms seem to be highly conserved from yeasts to mammals. In Saccharomyces cerevisiae, NPCs are constructed from ϳ30 different nucleoporins with a combined mass of ϳ50 MDa (26). The mammalian NPCs are much larger complexes (ϳ120 MDa) composed of ϳ80 different proteins (10). Although the structural organization of NPCs, the transport mechanism across the channels, and the function of individual nucleoporins have been extensively studied in yeast and vertebrates, very little is known about NPCs in plants.Recently, it was reported that the Arabidopsis thaliana proteins MOS3/SAR3 and SAR1 share high sequence similarities with human nucleoporins Nup96 and Nup160, respectively (25, 34). The putative nucleoporin MOS3/SAR3 was localized at the nuclear rim. The studies suggested that nucleocytoplasmic trafficking plays an important role in plant disease resistance, hormone signaling, and development (25,34). In the present report, we provide evidence that Arabidopsis nucleoporin AtNUP160/SAR1 controls nucleocytoplasmic transport of RNAs and plays important roles in seedling growth, flowering time regulation, and cold stress tolerance.Low temperature is one of...

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Targeted protein degradation (TPD) technology has drawn significant attention from researchers in both academia and industry. It is rapidly evolved as a new therapeutic modality and also a useful chemical tool in selectively depleting various protein targets. As most efforts focus on cytosolic proteins using PROteolysis TArgeting Chimera (PROTAC), LYsosome TArgeting Chimera (LYTAC) recently emerged as a promising technology to deliver extracellular protein targets to lysosome for degradation through the cation-independent mannose-6-phosphate receptor (CI-M6PR). In this study, we exploited the potential of the asialoglycoprotein receptor (ASGPR), a lysosomal targeting receptor specifically expressed on liver cells, for the degradation of extracellular proteins including membrane proteins. The ligand of ASGPR, triantennary N -acetylgalactosamine (tri-GalNAc), was conjugated to biotin, antibodies, or fragments of antibodies to generate a new class of degraders. We demonstrated that the extracellular protein targets could be successfully internalized and delivered into lysosome for degradation in liver cell lines specifically by these degraders. This work will add a new dimension to TPD with cell type specificity.

Histone deacetylase 6 (HDAC6) primarily catalyzes the removal of acetyl group from the side chain of acetylated lysine residues in cytoplasmic proteins such as αtubulin and HSP90. HDAC6 is involved in multiple diseaserelevant pathways. Based on the proteolysis targeting chimera strategy, we previously developed the first HDAC6 degrader by tethering a pan-HDAC inhibitor with cereblon (CRBN) E3 ubiquitin ligase ligand. We herein report our new generation of multifunctional HDAC6 degraders by tethering selective HDAC6 inhibitor Nexturastat A with CRBN ligand that can synergize with HDAC6 degradation for the antiproliferation of multiple myeloma (MM). This new class of degraders exhibited improved potency and selectivity for the degradation of HDAC6. After the optimization of the linker length and linking positions, we discovered potent HDAC6 degraders with nanomolar DC 50 and promising antiproliferation activity in multiple myeloma (MM) cells.