Using a combination of biochemical assays, structural modeling and molecular dynamics, it is shown that TTC33 directly recruits WDR61 and PHF5A to assemble into a trimeric core complex (TANC), which then forms distinct interactions with either UNG1/2 or SF3B–CCDC97.
Abstract
The functions of many human proteins remain unknown, highlighting major gaps in our understanding of cellular biology. TTC33 is an evolutionarily conserved tetratricopeptide repeat (TPR) protein expressed across human tissues, whose molecular role is not defined. Here we identify the TTC33 partners using comparative label-free mass spectrometry. The TTC33-associated network (TAN) comprises WDR61, CCDC97, UNG1/2, PP2A-B55α, PHF5A, and components of the SF3B U2 spliceosomal complex. Using a combination of biochemical assays, structural modeling and molecular dynamics we show that TTC33 directly recruits WDR61 and PHF5A to assemble into a trimeric core complex (TANC), which then forms distinct interactions with either UNG1/2 or SF3B–CCDC97. Somatic TTC33 mutations at key TANC interface residues reduce complex stability and weaken the interaction network. We further show that WDR61 stabilizes TTC33 by protecting it from proteolytic degradation. Loss of network components induces genomic instability and activates DNA damage markers, including γH2AX and p53 phosphorylation.
The interaction between mammalian enabled protein (Mena), an actin regulatory protein, and protein tyrosine phosphatase 1B (PTP1B) is critical for maintaining epidermal growth factor receptor (EGFR) signaling homeostasis and regulating cellular motility. Mena recruits PTP1B to activated EGFR in vivo, facilitating receptor dephosphorylation and limiting invasive signaling. However, overexpression of wild-type Mena or the invasive isoform, MenaINV, perturbs this regulatory mechanism by altering PTP1B localization and EGFR signaling dynamics. Here, we report the solution NMR structure of the unliganded Mena EVH1 domain, which contains a conserved aromatic triad (Y16, W23, F77) that forms the canonical polyproline-binding cleft. Building on prior evidence that the PTP1B polyproline region serves as an EVH1 ligand, we investigated the structural basis of the interaction between EVH1 and wild-type PTP1B. NMR chemical shift perturbation analyses using multiple PTP1B variants, including full-length and truncated constructs, revealed that binding affects not only the canonical EVH1 hydrophobic cleft but also residues on the opposite surface, suggesting an expanded interaction interface. Diffusion-ordered spectroscopy (DOSY) and size-exclusion chromatography further indicate that PTP1B variants containing the disordered C-terminal tail form more compact complexes with EVH1, consistent with a disorder-to-order transition. Complementary perturbation studies using [2H,15N] PTP1B demonstrate that EVH1 binding induces changes not only in the polyproline region but also across the phosphatase core, α7 helix, and C-terminal tail. Despite these structural perturbations, enzymatic assays show that EVH1 binding does not affect the catalytic activity of PTP1B, supporting a model in which Mena functions as a scaffold to spatially organize PTP1B within EGFR signaling complexes.
Lanette LaComb, Sean M. Cahill, J. Bonanno et al.· Biochemistry· 0 citations
Transcriptional programs regulated by the KDM5 family of chromatin-modifying proteins are dysregulated in cancer and intellectual disability (ID) disorders. To define the fundamental mechanisms by which KDM5 regulates disease-relevant gene expression, we use missense variants in the X-linked KDM5C gene associated with the ID disorder Claes-Jensen syndrome (also known as KDM5C-NDD). Here, we use Drosophila melanogaster to investigate the effects of KDM5A224T, equivalent to human KDM5CA77T, which affects a conserved residue outside the catalytic histone demethylase JmjC domain and alters both enzymatic and non-enzymatic activities. Quantifying levels of H3K4me3, the demethylase substrate of KDM5, in adult brains revealed that Kdm5A224T induced changes indistinguishable from those observed with a catalytically inactive allele. This effect was not due to reduced promoter recruitment of the variant KDM5A224T protein. Instead, TurboID studies demonstrate that KDM5A224T exhibits reduced proximity with proteins involved in promoter activity and chromatin remodeling. Together, these findings show that KDM5-dependent transcriptional regulation cannot be explained by demethylase activity alone and support altered chromatin regulatory interactions as a key mechanism underlying pathogenic KDM5 variants.
Melissa A. Castiglione, Matanel Yheskel, Aubrey A Siebels et al.· G3· 0 citations
Retrograde trafficking from endosomes to trans-Golgi network is essential for cellular homeostasis. While the WDR11-FAM91A1-C17orf75 (WFC) complex facilitates this process, its structural organization and the role of C17orf75 remain unclear. Here, we present cryo-EM structures of human WFC complex in monomeric and dimeric forms at 3.34 Å and 3.07 Å resolution, respectively. The WFC complex forms a dimer through the α-solenoid domains of WDR11. FAM91A1 serves as a central scaffold that interacts directly with both WDR11 and C17orf75, whereas WDR11 and C17orf75 have no direct contact. C17orf75 features an N-terminal longin-like domain and a C-terminal DENN-like domain and contains a positively charged groove that may cooperate with WDR11 in recognizing acidic-cluster-containing cargo proteins. Functional analyses demonstrate that C17orf75 is indispensable for the proper intracellular localization of the acidic-cluster-containing cargo proteins CI-MPR, KIAA0319L, and VAMP4. These findings establish C17orf75 as an integral component of the WFC complex in endosome-to-TGN trafficking.
A primary mechanism of SLC26A4 regulation in the inner ear and kidney and of SLC26A4 loss of function in Pendred syndrome and deafness DFNB4 is described and a atomistic model of the complete SLC26A4/ZBTB16/Cullin 3/RocI complex is provided in agreement with experimental results.
Florian Huber, E. Bernardinelli, Bassam G. Haddad et al.· Journal of Biomedical Scienc...· 0 citations
Bicaudal C Homolog 1 (BICC1) is a conserved RNA‐binding protein that, in mammals, has been primarily associated with polycystic kidney disease and renal organogenesis. However, its role in other disease contexts, including cancer, remains poorly understood. In this study, we characterized the BICC1 interactome, with emphasis on its dependence on RNA and the sterile alpha motif (SAM) domain, to identify novel biological processes associated with BICC1 function. Protein complexes were purified from HEK293T cells by co‐immunoprecipitation and analyzed by mass spectrometry. Notably, co‐immunoprecipitations performed in the presence of RNA yielded a larger number of interacting proteins, with 31 of 71 proteins (~43%) uniquely identified under RNA‐preserved conditions, highlighting the critical role of RNA in mediating BICC1 protein–protein interactions. Enriched proteins were predominantly associated with mRNA splicing, the PRMT5 methylosome complex, and membraneless organelles, such as biomolecular condensates. Consistent with these findings, immunofluorescence assays performed on stressed cells revealed the co‐localization of BICC1 with stress granule markers. Moreover, BICC1 interactions with PRMT5, STK38, PARP1, and IGF2BP1 were confirmed by immunoblotting.
Heloísa Monteiro do Amaral-Prado, G. A. Moreira, C. Santos et al.· FASEB bioAdvances· 0 citations
This review systematically synthesizes current knowledge regarding the multifaceted roles of YTHDC2 in disease progression, prognosis, and therapy, offering a comprehensive framework to guide future investigations.
Yan-Ying Hu, Qi Zhou, Ning Xu et al.· Cells· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
