Biology subjects

Tan, Z. Y.

Publications and source records attributed to Tan, Z. Y..

7 recordsLinked to original sources

bioRxiv2024

Discovery of molecular glues that bind FKBP12 and novel targets using DNA-barcoded libraries

Molecular glues are small molecules that engage their target and presenter proteins cooperatively. FKBP12 molecular glues (FK506 and rapamycin) were discovered several decades ago and have been used clinically, but our understanding of the breadth of FKBP12 molecular glues and targets has yet to be fully revealed. To identify novel targets of FKBP12 molecular glues, we constructed and screened a multi-million-member non-macrocyclic FKBP12-ligand DNA-encoded library using 25 structurally distinct proteins. Synthesis and validation of selected hits in biophysical and cell-based assays confirm FKBP12-dependent molecular-glue recruitment to bromodomain-containing protein 9 (BRD9) and quinoid dihydropteridine reductase (QDPR). One glue showed no measurable binding to QDPR alone but had appreciable binding in the presence of FKBP12 using either purified proteins or intact cells. The sites of recruitment were characterized with mutational analysis, competition-based methods and X-ray crystallography. The results of this study confirm that FKBP12-binding DELs can yield novel molecular glues generating highly selective FKBP12-target protein interactions.

biophysics↗

bioRxiv2024

Chemical tools to expand the ligandable proteome: diversity-oriented synthesis-based photoreactive stereoprobes

Chemical proteomics enables the global assessment of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, been limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically-defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these photo-stereoprobes interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible nanoBRET assays. Integrated phenotypic analysis and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and discovering and characterizing bioactive small molecules by cell-based screening.

biochemistry↗

bioRxiv2022

Diversity-oriented synthesis encoded by deoxyoligonucleotides

Diversity-oriented synthesis (DOS)is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors, derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

biochemistry↗

bioRxiv2021

Heterogeneous non-canonical nucleosomes predominate in yeast cells in situ

Nuclear processes depend on the organization of chromatin, whose basic units are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ (inside cells) resembles the canonical structure determined in vitro 25 years ago. Nucleosome structure in situ is otherwise poorly understood. Using cryo-ET and 3-D classification analysis of budding yeast cells, here we find that canonical nucleosomes account for less than 10% of total nucleosomes expected in situ. In a strain in which H2A-GFP is the sole source of histone H2A, class averages that resemble canonical nucleosomes both with and without GFP densities are found ex vivo (in nuclear lysates), but not in situ. These data suggest that the budding yeast intranuclear environment favors multiple non-canonical nucleosome conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average budding yeast nucleosomes DNA is partially detached in situ.

cell biology↗

bioRxiv2021

Endothelial alpha globin is a nitrite reductase

Small artery vasodilation in response to hypoxia is essential for matching oxygen supply to tissue oxygen demand. One source of hypoxic dilation via nitric oxide (NO) signaling is nitrite reduction by erythrocytic hemoglobin (2{beta}2). However, the alpha subunit of hemoglobin is also expressed in resistance artery endothelium and localized to myoendothelial junctions, a subcellular domain that contacts underlying vascular smooth muscle cells. We hypothesized that nitrite reduction mediated by endothelial alpha globin may occur at myoendothelial junctions to regulate hypoxic vasodilation. To test this concept, we created two novel mouse strains: one lacking alpha globin specifically in endothelium (EC Hba1{Delta}/{Delta}) and one where alpha globin is mutated such that its inhibitory association with endothelial NO synthase (eNOS) is prevented (Hba1WT/{Delta}36-39). In EC Hba1{Delta}/{Delta} or Hba1WT/{Delta}36-39 mice hemoglobin levels, hematocrit and erythrocyte counts were unchanged from littermate controls. Loss of the full alpha globin protein from the endothelium in the EC Hba1{Delta}/{Delta} model was associated with decreased exercise capacity and decreased intracellular nitrite utilization in hypoxic conditions. These effects were not seen in Hba1WT/{Delta}36-39 animals. Hypoxia induced vasodilation was decreased by 60% in isolated thoracodorsal arteries from EC Hba1{Delta}/{Delta}, while infusion of erythrocytes only partially rescued the dilatory response. Lastly, unlike other models where blood pressure is decreased, EC Hba1{Delta}/{Delta} blood pressure was not altered in response to hypoxia. Overall, we conclude that alpha globin in the resistance artery endothelium can act as a nitrite reductase to provide a local vasodilatory response to hypoxia.

physiology↗

bioRxiv2020

Macromolecular and biochemical changes of G0 fission yeast nuclei

When starved of nitrogen, fission yeast Schizosaccharomyces pombe cells enter a quiescent "G0" state with smaller nuclei and transcriptional repression. The genomics of S. pombe G0 cells has been well studied, but much of its nuclear cell biology remains unknown. Here we use confocal microscopy, immunoblots, and electron cryotomography to investigate the cytological, biochemical, and ultrastructural differences between S. pombe proliferating, G1-arrested, and G0 cell nuclei, with an emphasis on the histone acetylation, RNA polymerase II fates, and macromolecular complex packing. Compared to proliferating cells, G0 cells have lower levels of histone acetylation, nuclear RNA polymerase II, and active transcription. The G0 nucleus has similar macromolecular crowding yet fewer chromatin-associated multi-megadalton globular complexes. Induced histone hyperacetylation in G0 results in cells that have larger nuclei and therefore less compact chromatin. However, these histone-hyperacetylated G0 cells remain transcriptionally repressed with similar nuclear crowding. Canonical nucleosomes - those that resemble the crystal structure - are rare in proliferating, G1-arrested, and G0 cells. Our study therefore shows that extreme changes in nucleus physiology are possible without extreme reorganisation at the macromolecular level. SummaryWe use multiple cell-biological techniques to compare proliferating and quiescent G0 fission yeast cell nucleus transcription, histone acetylation, macromolecular packing.

cell biology↗

bioRxiv2017

Cryo-ET reveals nucleosome reorganization in condensed mitotic chromosomes in vivo

Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription, but the details remain unclear. Here, we use cryo-electron tomography to determine the 3-D arrangement of nucleosomes and other large nuclear features in frozen-hydrated fission-yeast cells. Nucleosomes can form irregular clusters in both interphase and mitotic cells, but they are smaller than expected for Hi-C domains. The nucleosomes are co-mingled with two features: nucleosome-free pockets and megadalton-sized \"megacomplexes\". Compared to interphase, the nucleosomes in mitotic chromosomes pack into slightly larger clusters. However, nearest-neighbor distance analysis reveals that mitotic nucleosome clusters have the same internal packing density as in interphase. Furthermore, mitotic chromosomes contain fewer megacomplexes. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: most genes are repressed but a subset is upregulated.

cell biology↗