Aug 2026· Journal of Biomedical Sciences· Vol 33· 0 citations· 100 references
Medicine
TL;DR
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.
Abstract
Pathogenic sequence alterations in the SLC26A4 gene, which encodes the solute carrier SLC26A4/pendrin, lead to Pendred syndrome and non-syndromic autosomal recessive deafness type B4 (DFNB4), two of the most common forms of hearing loss worldwide. Many pathogenic SLC26A4 protein variants exhibit reduced cellular levels due to ubiquitin-proteasome system (UPS)-mediated degradation, and UPS inhibition rescues their plasma membrane expression and ion transport function. However, the underlying molecular mechanisms remain unclear and may involve interactions with novel molecular partners. A candidate SLC26A4 protein partner was found by a yeast two-hybrid screening. The biological significance of this interaction has been studied by immunohistochemistry and co-localization in the mouse inner ear and kidney, co-immunoprecipitation of endogenous and recombinant proteins, Liquid Chromatography-Tandem Mass Spectrometry, and Fluorescence Resonance Energy Transfer. We identified the zinc finger and BTB domain-containing protein ZBTB16 as a novel SLC26A4-interacting partner. ZBTB16 co-localized with SLC26A4 in the outer sulcus and spiral prominence epithelial cells of the mouse cochlea and in the apical membrane of non-alpha non-beta intercalated cells of the distal nephron. ZBTB16 was found to be part of a ubiquitin-ligase complex comprising the scaffold protein Cullin 3 and the ubiquitin ligase RocI, and to bind with its C-terminal zinc finger region a unique amino acid sequence within the C-terminal Sulfate Transporter and Anti-Sigma factor Antagonist (STAS) domain of SLC26A4. This direct molecular interaction leads to increased site- and variant-specific ubiquitination and accelerated degradation of SLC26A4. Finally, using AI-based structure prediction, we provide an atomistic model of the complete SLC26A4/ZBTB16/Cullin 3/RocI complex in agreement with our experimental results. These findings describe a primary mechanism of SLC26A4 regulation in the inner ear and kidney and of SLC26A4 loss of function in Pendred syndrome and deafness DFNB4, and identify potential novel molecular targets for therapeutic intervention.
The carboxyl terminus of Hsp70-interacting protein (CHIP, encoded by STUB1) combines co-chaperone and E3 ubiquitin ligase activities to regulate protein quality control. Heterozygous mutations in STUB1 cause spinocerebellar ataxia type 48 (SCA48), a progressive cerebellar ataxia with variable extrapyramidal and cognitive features. To understand the molecular basis of this variability, we systematically analyzed 13 SCA48-associated variants spanning the TPR and U-box domains through recombinant protein biochemistry and cellular models. TPR variants retained intrinsic ligase activity but showed significantly reduced HSP70 binding, impaired substrate ubiquitination, and decreased stability. Conversely, U-box variants abolished ligase function, promoted the formation of high-molecular-weight oligomers, and often increased CHIP levels while only partially impairing co-chaperone activity. Many mutants displayed temperature-sensitive defects and defective stress-induced nuclear translocation. Principal component analysis revealed distinct biochemical clustering specific to each domain. RNA-seq following STUB1 knockdown modeled CHIP insufficiency and showed preserved HSF1-dependent transactivation, but loss of CHIP's capacity to amplify ubiquitination, chaperone function, and stress-related transcriptional programs. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical signatures to upper motor neuron involvement and U-box-like profiles to prominent dysarthria. Overall, these data indicate that SCA48 results from domain-specific disruption of CHIP's dual functions, producing varying degrees of CHIP insufficiency and/or gain-of-toxic effects that together contribute to the phenotypic diversity observed across patients. This work refines the mechanistic framework for SCA48 pathogenesis and highlights strategies for therapeutic modulation of residual CHIP activity.
S. Altinok, E. Paulakonis, Michael F. Almeida et al.· Journal of Biological Chemis...· 0 citations
Alu elements are primate-specific retrotransposons known to mediate structural genomic alterations associated with neurological disorders, yet their underlying disease mechanism remains incompletely understood. Here, we leveraged human pluripotent stem cell-derived cortical organoids and xenografts to model an Alu-mediated exon 17 deletion in the SPAST gene, a frequent cause of hereditary spastic paraplegia (SPG4) with dementia. This deletion generates fusion transcripts between SPAST and the downstream gene SLC30A6, resulting in reduced expression of ZnT6, the Golgi-resident zinc transporter encoded by SLC30A6. In SPASTΔe17 organoids, we detected pronounced cytosolic zinc accumulation, Golgi fragmentation, lipid dysregulation, and increased aggregation of Aβ and ubiquitin. These phenotypes were further exacerbated in xenograft models, concomitant with ubiquitin-positive inclusions resembling those found in patients’ brains. Knockdown of SLC30A6 in control organoids recapitulated the neurodegenerative features, supporting a critical role of ZnT6 in maintaining neuronal homeostasis. Intervention with TPEN, a zinc-specific chelator, or a GRASP55-blocking antibody to prevent Golgi fragmentation significantly restored Golgi integrity and reduced Aβ levels. Notably, immunohistochemistry revealed heterogeneous ZnT6 expression in AD brains; however, both ZnT6-low and ZnT6-high cases consistently exhibited elevated p-GM130, a marker of Golgi fragmentation. Finally, we detected a SPAST–SLC30A6 chimeric transcript in brain tissue from a sporadic AD patient. Together, our findings identify a primate-specific cascade linking Alu-mediated genomic rearrangements to zinc dyshomeostasis and Golgi pathology and suggest that dysregulation of the ZnT6–Golgi axis may represent a vulnerability and therapeutic target in structural variant–driven neurodegeneration and possibly in a subset of sporadic cases.
Youngsun Lee, Onju Ham, Hana Lee et al.· Signal Transduction and Targ...· 0 citations
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.
R. Tomecki, Małgorzata Drabko, Małgorzata Siek et al.· Nature Communications· 0 citations
Introduction Autosomal recessive Parkinson’s disease (ARPD) arises from impaired mitophagy due to dysfunction of the PINK1/Parkin pathway, where PINK1-mediated phosphorylation of ubiquitin at Ser65 is essential for pathway activation. However, experimental limitations obscure the effects of disease-associated mutations on intrinsically disordered regions (IDRs) and post-translational modification (PTM) dynamics. Methods An integrated computational pipeline was employed to screen 825 PINK1 missense variants, identifying two high-confidence deleterious mutations, T313M and L347P, within the kinase domain. Variant prioritization was complemented by conserved residue, IDR, and PTM analyses, followed by protein–protein docking, molecular dynamics simulations, MM/PBSA binding free-energy calculations, principal component analysis (PCA), and dynamic cross-correlation matrix (DCCM) analysis. Results T313M overlapped a conserved phosphorylation site, whereas L347P mapped to conserved active-site residues, with complementary support from IDR analysis. Docking analysis revealed a progressive reduction in binding affinity from the wild type (–88.4 ± 8.2) to L347P (–81.9 ± 3.4) and T313M (–77.4 ± 4.9), accompanied by decreased electrostatic stabilization (–357.6 → –260.6 → –235.5 kcal/mol) and buried surface area (1741.6 → 1624.1 → 1547.4 Å2). Molecular dynamics simulations demonstrated that T313M produced the greatest structural and dynamic perturbations, whereas L347P induced moderate destabilization with increased solvent exposure. Although MM/PBSA analysis indicated broadly comparable binding energetics across all systems, PCA and DCCM analyses revealed increased conformational flexibility and altered residue communication in the mutant complexes, particularly T313M. Discussion These findings establish a mechanistic link between mutation-induced structural dynamics and impaired PINK1–ubiquitin recognition at Ser65, providing a mutation-specific framework for understanding early mitophagy impairment in ARPD and supporting future molecular assessment and targeted therapeutic development.
Zhiguang Jia, B. Malik, Deborah Vincent et al.· Frontiers in Molecular Neuro...· 0 citations
As a key N6-methyladenosine (m6A)-binding protein, YT521-B Homology (YTH) Domain-Containing Protein 2 (YTHDC2) plays a central role in the epitranscriptomic regulatory network. This protein specifically recognizes and binds to m6A modification sites on RNA molecules through its highly conserved YTH domain. This recognition exhibits high selectivity and affinity, thereby enabling precise control over the fate of target RNAs. At the molecular level, YTHDC2 is widely involved in various stages of the RNA life cycle, including core biological processes such as RNA splicing and processing, nuclear–cytoplasmic transport, translational efficiency regulation, and RNA decay. In recent years, accumulating evidence indicates that YTHDC2 participates in a variety of pathophysiological processes in an m6A-dependent manner. However, the robustness of evidence regarding YTHDC2 is heterogeneous across disease contexts. While certain pathologies are supported by rigorous mechanistic validation, others rely primarily on expression correlations or bioinformatic analyses. 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
β-propeller protein-associated neurodegeneration (BPAN) is the most prevalent subtype of neurodegeneration with brain iron accumulation (NBIA) and is caused by mutations in the WDR45 gene encoding the autophagy-related protein WIPI4. However, many WDR45 missense variants remain classified as variants of uncertain significance (VUS), highlighting the need for reliable functional assays to support their clinical interpretation. In this study, we identified the yeast ortholog of WDR45 and established a Saccharomyces cerevisiae-based functional complementation assay to assess the pathogenicity of WDR45 variants. We first showed that deletion of the β-propellers that bind polyphosphoinositides (PROPPIN)-encoding genes ATG18 or HSV2 causes mitochondrial dysfunction and impaired respiratory growth. Human WDR45/WIPI4 specifically rescued the respiratory defect of the atg18Δ strain, whereas WDR45B/WIPI3 complemented the hsv2Δ phenotype, establishing yeast Atg18 as the closest functional counterpart of WIPI4, thus clarifying PROPPIN orthology. We then evaluated a panel of WDR45 variants and found that benign variants restored normal growth, whereas truncating and pathogenic missense variants failed to complement the atg18Δ phenotype, validating the assay for functional variant classification. Finally, we analyzed several VUS identified in patients with clinically compatible BPAN and obtained functional evidence supporting their pathogenicity. Overall, our study establishes a simple, robust, and scalable yeast model that enables functional interpretation of WDR45 variants and improves molecular diagnosis of BPAN.
Vincent Morin, C. Durand, C. Angelini et al.· International Journal of Mol...· 0 citations