Biology subjects

Kiselar, J.

Publications and source records attributed to Kiselar, J..

3 recordsLinked to original sources

bioRxiv2025

Structural and functional basis of PU.1-BAF interaction enables targeting of lineage-specific transcription

Chromatin remodeling complexes like BAF finely regulate transcriptional programs by working in concert with transcription factors. However, evidence is lacking as to whether TFs interact directly with BAF and if so, what the mechanistic and structural principles governing these critical interactions are. Here, we establish direct engagement between a crucial and therapeutically relevant full-length human TF, PU.1 (SPI1), and BAF. Within this 1MDa+ complex, we precisely map the binding site of PU.1 to a YEATS-like domain on BAF60A and elucidate the structure of the PU.1-BAF60A complex. This work reveals that upon binding to BAF, a disordered region within the TF adopts a helical conformation, and that disruption of this functionally critical interface via knockdown abrogates the ability of PU.1 to rescue cell viability. To explore the druggability of TF-BAF protein-protein interactions (PPIs), we conducted a high-throughput screen that identified small molecules capable of disrupting the PU.1-BAF60A PPI by binding to BAF60A. Co-crystal structures reveal distinct compound binding modes that converge on a critical PU.1-BAF60A interaction hotspot. These findings define, for the first time, the structural interface between a human TF and a chromatin remodeling complex and establish a platform that enables the targeting of these interactions, a novel mechanism in cancer therapeutics.

biochemistry↗

bioRxiv2021

GABARAP membrane conjugation sequesters the FLCN-FNIP tumor suppressor complex to activate TFEB and lysosomal biogenesis

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that during single membrane ATG8 conjugation (SMAC), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal homeostasis and capacity. GABARAP directly binds to a novel LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex with picomolar affinity and regulates its relocalization to these GABARAP-conjugated membrane compartments. This disrupts the regulation of RagC/D by the FLCN/FNIP GAP complex, resulting in impaired mTOR-dependent phosphorylation of TFEB without changing mTOR activity towards other substrates. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a universal molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

cell biology↗

bioRxiv2018

Metastable contacts and structural disorder in the estrogen receptor transactivation domain

The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins (IDPs), mediates the receptors transactivation function to regulate gene expression. However, an accurate molecular dissection of NTDs structure-function relationships remains elusive. Here, using small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), circular dichroism, and hydrogen exchange mass spectrometry, we show that NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion upon chemical denaturation. By combining SAXS, hydroxyl radical protein footprinting and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map that reveals metastable regional and long-range contacts, including interactions between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) NMR that mutations near residue I33 alter 19F chemical shifts at residue S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of IDPs structure-function relationship, and how specific metastable contacts mediate critical functions of disordered proteins.\n\nHighlightsO_LIA compact disorder is observed for the N-terminal domain (NTD) of estrogen receptor\nC_LIO_LIMulti-technique modeling elucidates the NTD ensemble structures\nC_LIO_LIEnsemble-based contact map reveals metastable contacts between I33 and S118\nC_LIO_LI19F-NMR data validate the proposed I33-S118 contact in the IDP\nC_LI

biophysics↗