Roy Merin Vellooparambil, Neal Scott J, Pignoni Francesca
Department of Biochemistry and Molecular Biology, Upstate Medical University, Syracuse, New York, United States of America.
Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, New York, United States of America.
PLoS One. 2025 Jun 4;20(6):e0325326. doi: 10.1371/journal.pone.0325326. eCollection 2025.
The evolutionarily conserved Lipocalin-Interacting Membrane Receptor (LIMR) family (InterPro: IPR006876) consists of transmembrane (TM) proteins characterized by 9 TM domains (TMDs). Their reported biological functions are diverse and remain poorly understood. In previous work, we showed that the fly family member Lilipod (Lili) impacts biological processes regulated by the fly BMP/TGF-β ligand Decapentaplegic (Dpp), including germline stem cell self-renewal in the Drosophila ovary, dorsal closure during embryonic development and wing vein formation at the pupal stage. Based on this genetic evidence, Lili directly or indirectly enhances bone morphogenetic protein (BMP) signaling. In the ovary, Lili functions between the activated type I BMP receptor and the SMAD intracellular transducer. To gain insight into Lili function at the cellular and molecular levels, we probed the functional significance of its largest intracellular loop, Intracellular Loop 3 (ICL3). Through mutational analysis, we mapped sequences critical for Lili function in vivo to the evolutionarily conserved regions of ICL3. Additionally, we showed that fly-human chimeric proteins in which Lili ICL3 is replaced with the ICL3 of its human homologs, LMBR1 and LMBR1L, can rescue lili null-mutant phenotypes. Using ICL3 as bait in an unbiased Yeast 2-Hybrid (Y2H) screen, we identified putative interactors, including the BMP signaling cascade components Mad, Sara, Nup93 and Nup358, and further Y2H analyses identified distinct regions on ICL3 as potentially important for protein binding. Taken together, our work has identified ICL3 as a region that is critical for Lili protein function, most likely via its mediation of protein-protein interactions (PPIs).
进化上保守的脂钙素相互作用膜受体（LIMR）家族（InterPro：IPR006876）由具有9个跨膜结构域（TMD）的跨膜（TM）蛋白组成。它们已报道的生物学功能多种多样，目前仍知之甚少。在之前的研究中，我们发现果蝇家族成员Lilipod（Lili）会影响由果蝇BMP/TGF-β配体Decapentaplegic（Dpp）调控的生物学过程，包括果蝇卵巢中的生殖系干细胞自我更新、胚胎发育期间的背侧闭合以及蛹期的翅脉形成。基于这一遗传学证据，Lili直接或间接增强骨形态发生蛋白（BMP）信号传导。在卵巢中，Lili在活化的I型BMP受体和SMAD细胞内转导器之间发挥作用。为了深入了解Lili在细胞和分子水平上的功能，我们探究了其最大的细胞内环——细胞内环3（ICL3）的功能意义。通过突变分析，我们将Lili在体内发挥功能的关键序列定位到ICL3的进化保守区域。此外，我们还表明，用其人类同源物LMBR1和LMBR1L的ICL3替换Lili ICL3的果蝇-人类嵌合蛋白可以挽救lili无效突变体表型。在无偏向性的酵母双杂交（Y2H）筛选中，以ICL3作为诱饵，我们鉴定出了推定的相互作用蛋白，包括BMP信号级联成分Mad、Sara、Nup93和Nup358，进一步的Y2H分析确定了ICL3上不同区域对蛋白质结合可能很重要。综上所述，我们的研究确定ICL3是对Lili蛋白功能至关重要的区域，很可能是通过其介导的蛋白质-蛋白质相互作用（PPI）来实现的。
