Biology subjects

Gonzalez, D. P.

Publications and source records attributed to Gonzalez, D. P..

3 recordsLinked to original sources

bioRxiv2025

A yeast model reveals the SMAD3 nuclear import mechanism

TGF-{beta} signaling is important for patterning tissues during embryonic development and relies on the nuclear import of SMADs. Basal SMAD nuclear import mechanisms remain to be fully understood and could benefit from advances in our understanding of nuclear transport machinery. To assess the molecular determinants of SMAD nuclear transport, we take advantage of the unicellular S. cerevisiae model that lacks the TGF-{beta} signaling complexity of vertebrates but has conserved nuclear transport machinery. In this minimal system, we find that the steady-state distribution of SMAD2/3, SMAD4, and SMAD1/5 are Ran-dependent and either accumulate in, or are depleted from the nucleus. Conditionally inhibiting each of the karyopherin/importins demonstrates that SMAD3 is imported by orthologues of importin-{beta}, importin-7 and importin-8. The previously defined nuclear localization signal is insufficient to confer nuclear import. Instead, our data suggest the entire MH1 domain may act as an importin binding surface. Deleting a portion of this domain impairs SMAD3 function in Xenopus and SMAD3 nuclear enrichment in HEK293T cells. Thus, the yeast platform provides an efficient strategy to illuminate the nuclear transport mechanisms of embryonic signaling effectors.

developmental biology↗

bioRxiv2021

Kap-β2/Transportin mediates β-catenin nuclear transport in Wnt signaling

Wnt signaling is essential for many aspects of embryonic development including the formation of the primary embryonic axis. In addition, excessive Wnt signaling drives multiple diseases including cancer highlighting its importance for disease pathogenesis. {beta}-catenin is a key effector in this pathway that translocates into the nucleus and activates Wnt responsive genes. However, due to our lack of understanding of {beta}-catenin nuclear transport, therapeutic modulation of Wnt signaling has been challenging. Here, we took an unconventional approach to address this long-standing question by exploiting a heterologous model system, the budding yeast Saccharomyces cerevisiae, which contains a conserved nuclear transport machinery. In contrast to prior work, we demonstrate that {beta}-catenin accumulates in the nucleus in a Ran dependent manner, suggesting the use of a nuclear transport receptor (NTR). Indeed, a systematic and conditional inhibition of NTRs revealed that only Kap104, the orthologue of Kap-{beta}2/Transportin-1 (TNPO1), was required for {beta}-catenin nuclear import. We further demonstrate direct binding between TNPO1 and {beta}-catenin that is mediated by a conserved amino acid sequence that resembles a PY NLS. Finally, using Xenopus secondary axis and TCF/LEF reporter assays, we demonstrate that our results in yeast can be directly translated to vertebrates. By elucidating the NLS in {beta}-catenin and its cognate NTR, our study provides new therapeutic targets for a host of human diseases caused by excessive Wnt signaling. Indeed, we demonstrate that a small chimeric peptide designed to target TNPO1 can reduce Wnt signaling as a first step towards therapeutics.

developmental biology↗

bioRxiv2018

CBD-1 scaffolds two independent complexes required for eggshell vitelline layer formation and egg activation in C. elegans

Metazoan eggs have a specialized coat of extracellular matrix that aids in sperm-egg recognition. The coat is rapidly remodeled after fertilization to prevent polyspermy and establish a more permanent barrier to protect the developing embryo. In nematodes, this coat is called the vitelline layer, which is remodeled into the outermost layer of a rigid and impermeable eggshell. We have identified three key components of the vitelline layer structural scaffold - PERM-2, PERM-4 and CBD-1, the first such proteins to be described in the nematode C. elegans. CBD-1 recruited PERM-2 and PERM-4 to the nascent vitelline layer via two N-terminal chitin-binding domains. After fertilization, all three proteins redistributed from the zygote surface to the outer eggshell. Depletion of PERM-2 and PERM-4 from the scaffold led to a porous vitelline layer that permitted soluble factors to leak through the eggshell and resulted in embryonic death. In addition to its role in vitelline layer assembly, CBD-1 is also known to scaffold a protein complex required for fertilization and egg activation (EGG-1-5/CHS-1/MBK-2). We found the PERM complex and EGG complex to be structurally and functionally independent, and regulated through distinct domains of CBD-1. CBD-1 is thus a multifaceted regulator that promotes distinct aspects of vitelline layer assembly and egg activation. In sum, our findings identify the first vitelline layer components in nematodes, and provide a foundation through which to explore both conserved and species-specific strategies used by animals to build protective barriers following fertilization.

developmental biology↗