Biology subjects

Ortiz-Pacheco, J.

Publications and source records attributed to Ortiz-Pacheco, J..

3 recordsLinked to original sources

bioRxiv2026

Integrated Interactomics Reveals Novel Protein Associations: The FOXA1-PBX1 Complex as a Case Study

Protein-protein interactions (PPIs) are fundamental to cellular signaling networks, yet many remain undetected due to technical limitations of individual affinity purification approaches. To address this, we systematically mapped the interaction landscapes of six regulatory proteins involved in cell proliferation, immunity, and inflammation, including three transcription factors (TFs) and three kinases. We implemented an integrated proteomics workflow that combined four complementary affinity purification strategies: native immunoprecipitation, two crosslinking-assisted capture methods, and proximity labeling. Combining these approaches revealed distinct yet overlapping interaction profiles, uncovered numerous previously unreported interactors not reliably detected by individual methods, and robustly recovered known interactions while substantially extending PPI networks. Despite method-specific differences at the protein level, functional enrichment analyses showed strong convergence on coherent biological pathways. Biochemical approaches validated most of the previously unreported interactions, including putative weak and transient complexes stabilized by crosslinking. Functional assays revealed a previously unrecognized physical interaction between FOXA1 and PBX1 TFs and demonstrated their cooperative regulation of transcriptional programs and cell fitness in estrogen receptor (ER) positive breast cells. We propose that the FOXA1-PBX1 complex could represent a higher-order regulatory node integrating chromatin accessibility and ER-driven transcriptional output. HIGHLIGHTSO_LIComplementary affinity purification strategies uncover putative weak and transient protein-protein interactions C_LIO_LIFunctional pathway convergence validates biologically coherent interactome expansion C_LIO_LIBiochemical validations confirm unreported interactions C_LIO_LIFunctional validation studies identify a FOXA1-PBX1 pioneer factor complex that regulates estrogen receptor transcriptional programs C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/738938v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@193cc1borg.highwire.dtl.DTLVardef@3d068dorg.highwire.dtl.DTLVardef@791d5corg.highwire.dtl.DTLVardef@1769ce7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Bound for the nucleus: defining the molecular principles of cargo selection by importin 9

Active nuclear transport of proteins enables essential nuclear processes, such as genome packaging, transcription, splicing, ribosome biogenesis, and DNA repair. To facilitate proper sorting between cytoplasmic and nuclear compartments, the importin class of chaperone proteins transports hundreds of proteins through hydrophobic nuclear pores. While some importins recognize cargos via classical nuclear localization signals (cNLSs), there is not a comprehensive understanding of binding specificity determinants for those that appear to recognize other cargo features. Here we subject one such importin, IPO9, which is known to import H2A-H2B, TFIIB, actin, and the proteasome into the nucleus without cNLS binding, to a detailed analysis of its full set of cargos and the structural elements of IPO9 that confer their recognition. Through cytoplasmic immunoprecipitation followed by mass spectrometry (IP-MS), we stringently and reproducibly identify 79 bona fide IPO9-bound cargos, including 20 previously validated cargos. With this comprehensive cargo list, we find that IPO9 does not appear to use cNLSs, nor any other linear peptide motif, to identify and bind cargos. Unbiased oxidative footprinting of extracted IPO9*cargo complexes reveals that both the inner cavity of IPO9 and the unstructured loops protruding from the main body of the importin are protected by bound cargo, indicative of cargo competition for limiting IPO9 capacity. Guided by these data and evolutionary and phosphoproteomics insights, we employ a systematic IP-MS approach with loop perturbations to define how each element contributes to selective binding of subsets of the whole cargo cohort. These data suggest the H8 and H18-19 loops both specifically mediate IPO9 cargo-recognition by favorable enthalpic contacts. Additionally, these loops, as well as H7, appear to preclude binding of a secondary set of potential cargo to IPO9 which may normally be repelled by them or outcompeted by cognate cargo binding for which the loops provide attractive contacts. We define the continuum of cargo-release factor RanGTP sensitivity for our full cargo set, noting orders of magnitude range of sensitivity that argues that additional release factors may be necessary for efficient unloading in the nucleus. These experiments provide structure-function insight into importin binding specificity dictated by structural elements that recruit and/or restrict protein-protein interactions. Our approach of targeted mutations in cellular contexts coupled to quantitative proteomics affords a thorough biochemical dissection of the discrimination principles undergirding this unique molecular recognition problem of numerous-yet-specific binding events between importins and their many distinct cargos.

molecular biology↗

bioRxiv2025

GMCL1 Controls 53BP1 Stability and Modulates Paclitaxel Sensitivity in Cancer

Mitotic surveillance pathways monitor the duration of mitosis (M phase) in the cell cycle. Prolonged M phase, caused by spindle attachment defects or microtubule-targeting drugs triggers formation of the ternary "mitotic stopwatch pathway" complex (MSP) consisting of 53BP1, USP28, and p53. This complex stabilizes p53, leading to cell cycle arrest or apoptosis in daughter cells. In cancers that are resistant to paclitaxel, a microtubule-targeting agent, cells bypass mitotic surveillance activation, allowing unchecked proliferation, although the underlying mechanisms remain poorly understood. Here, we identify GMCL1 as a key negative regulator of MSP signaling. We show that 53BP1 physically interacts with GMCL1, but not its paralog GMCL2, and we map their interaction domains. CRL3GMCL1 functions as a ubiquitin ligase that targets 53BP1 for degradation during the M phase, thereby reducing p53 accumulation in daughter cells. Depletion of GMCL1 inhibits cell cycle progression upon release from prolonged mitotic arrest, a defect that is rescued by co-silencing 53BP1 or USP28. Moreover, GMCL1 depletion sensitizes cancer cells to paclitaxel in a p53-dependent manner. Together, our findings support a model in which dysregulated CRL3GMCL1-mediated degradation of 53BP1 prevents proper MSP function, leading to p53 degradation and continued proliferation. Targeting GMCL1 may therefore represent one possible avenue for addressing paclitaxel resistance in cancer cells with functional p53.

cell biology↗