Biology subjects

Sabatini, D. M.

Publications and source records attributed to Sabatini, D. M..

4 recordsLinked to original sources

bioRxiv2018

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (\"MITO-Tag\") for the fast isolation of mitochondria from cultured cells to now generate \"MITO-Tag Mice.\" Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

molecular biology↗

bioRxiv2018

Identification and characterization of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

AbstractNitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

biochemistry↗

bioRxiv2018

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a significant improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling both in vitro and in vivo assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells.

cancer biology↗

bioRxiv2017

Juxtacap Nucleotide Sequence Modulates eIF4E Binding and Translation

mRNA translation is an energetically costly activity required for almost all biological processes. The multiprotein complex eIF4F, which bridges the 5 cap and the polyA tail through eIF4E and eIF4G, respectively, is necessary for efficient translation initiation of most mRNAs and is an important target of translational control. Previous work suggests that cap-proximal nucleotides can modulate eIF4E binding to mRNAs, but the effect of specific cap-proximal nucleotide sequences on eIF4E recruitment and the ultimate consequences for translation remain unknown. Using RNA Bind-n-Seq on a model 5 UTR, we systematically identify eIF4E-intrinsic cap-proximal nucleotide binding preferences. mRNAs with highly-bound motifs are translated well in a cell-free system, whereas those with low-eIF4E-binding motifs are not. However, eIF4E juxtacap motif preferences do not dictate the ribosome occupancy of endogenous mRNAs in cells, suggesting that the effect of juxtacap sequence on eIF4E binding and translation is mRNA-context-dependent. Accordingly, a single downstream point mutation that disrupts a predicted base pair with a preferred juxtacap nucleotide increases translation. The juxtacap sequence is a previously unappreciated determinant of eIF4E recruitment to mRNAs, and we propose that differences in mRNA 5 end accessibility defined by the juxtacap sequence are important for establishing translational efficiency.

biochemistry↗