Biology subjects

Fiedorczuk, K.

Publications and source records attributed to Fiedorczuk, K..

3 recordsLinked to original sources

bioRxiv2023

Structural basis for CFTR inhibition by CFTRinh-172

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. Whereas activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease (ADPKD). Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh- 172 to 2.7 [A] resolution by cryogenic electron microscopy (cryo-EM). We observe that CFTRinh- 172 binds inside the pore near transmembrane helix 8 (TM8), a critical structural element that links ATP hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed and the pore is blocked from the extracellular side of the membrane. Single molecule fluorescence resonance energy transfer (smFRET) experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain (NBD) dimerization. Together, these data show that CFTRinh-172 acts as both a pore blocker and a gating modulator, setting it apart from typical ion channel inhibitors. The dual functionality of CFTRinh-172 reconciles previous biophysical observations and provides a molecular basis for its activity. Significance statementThe pathogenesis of secretory diarrhea and autosomal dominant polycystic kidney disease involves hyperactivation of the CFTR ion channel. CFTR inhibitors, including the small-molecule CFTRinh-172, have been developed as therapeutic candidates to treat these diseases. This study offers a structural understanding of CFTRinh-172s mode of action, clarifying its dual inhibitory role as both a pore blocker and gating modulator. The molecular description of how CFTRinh-172 interacts with CFTR provides a structural foundation to its specificity and efficacy. Furthermore, the observation that CFTR inhibitors and potentiators both interact with TM8 strengthens the notion that this helix serves as an allosteric link between the ATPase site and the channel gate, and is therefore a hotspot for pharmacological modulation.

biophysics↗

bioRxiv2023

Universal recording of cell-cell contacts in vivo for interaction-based transcriptomics

Cellular interactions are essential for tissue organization and functionality. In particular, immune cells rely on direct and usually transient interactions with other immune and non-immune populations to specify and regulate their function. To study these "kiss-and-run" interactions directly in vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the cellular partners of regulatory T cells in steady state, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) and find evidence of stepwise acquisition of the ability to interact with IECs as CD4+ T cells adapt to residence in the intestinal tissue. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

immunology↗

bioRxiv2021

Mechanism of CFTR correction by type I folding correctors

Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the {Delta}F508 mutant and are widely used to treat patients. However, their mechanism of action is unknown. Here we present cryo-electron microscopy structures of CFTR in complex with two FDA-approved correctors: lumacaftor and tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered {Delta}F508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its pre-mature degradation, and thereby allosterically rescue a large number of disease-causing mutations.

biophysics↗