Biology subjects

Beekman, J.

Publications and source records attributed to Beekman, J..

7 recordsLinked to original sources

bioRxiv2026

Spike and host glycan determinants of HKU1 airway tropism

Human coronavirus HKU1 comprises two distinct serotypes, A and B, whose spike proteins are substantially divergent. Here, we show that spikes from both serotypes preferentially bind 9-O-acetylated 2,8-linked disialosides. Cryo-electron microscopy of the B-type N5 spike reveals a conserved extended binding site in domain S1A that accommodates both the terminal and penultimate sialic acid residues, with interactions involving the penultimate residue substantially enhancing binding. Spike N-glycan processing modulates affinity and linkage selectivity; glycans flanking the binding pocket offer a plausible structural basis for these effects. In contrast to the HKU1-A spike, which adopts open S1B-up conformations upon ligand binding, the N5 apo structure showed that the ligand-binding site was already formed and the e1 relay element register-shifted in most protomers. Nevertheless, we detected neither spontaneous opening nor a transition to an S1B-up state following ligand binding. These findings, obtained with a minimally modified ectodomain, differ from recent reports of ligand-independent opening. Molecular dynamics simulations indicated that membrane-embedded GT3, but not GD3, presents its glycan chain in a geometry compatible with S1A-mediated engagement. Concordantly, in human nasal epithelial cultures cell surface GT3-like O-acetylated trisialoside glycotopes were detected in ciliated cells, linking their cell-type-specific presentation to HKU1 tropism.

microbiology↗

bioRxiv2026

Structure of the mammalian ciliary transition zone microtubule doublet

The ciliary transition zone gates bidirectional protein trafficking to maintain the specialized ciliary proteome using microtubule doublets as a scaffold. While ciliary axonemal doublets are well-characterized, the molecular architecture of the transition zone doublet remains elusive. Here, we report the structure of the mammalian transition zone doublet from bovine tracheal cilia using cryo-electron tomography at 4.7-5.0 [A] resolution. The transition zone doublet is a structurally independent segment defined by an 8 nm-periodic arrangement of unique microtubule-inner proteins (MIPs) and microtubule-associated proteins (MAPs). We identify the calcium-binding protein CAPSL as a lumenal MIP that forms a pseudo-helical spiral and stabilizes microtubules in vitro. Furthermore, a dense MAP network on the A-microtubule surface clashes with intraflagellar transport (IFT) motor binding sites, suggesting anterograde IFT is directed to the B-microtubule for potential regulation by MAP9. Our work provides a structural framework for understanding gated ciliary transport and transition zone-linked human ciliopathies.

Cell Biology↗

bioRxiv2025

Distinct heterozygous TTC7A missense variants lead to different intestinal epithelial phenotypes in pediatric IBD

Pathogenic mutations in Tetratricopeptide repeat domain 7A (TTC7A) result in gastrointestinal and immunological disorders of which the pathobiology is not fully understood. Previous case reports indicate that TTC7A plays an important role in preserving intestinal epithelial integrity, but thus far only few variants have been investigated and it is unclear if different variants exert the same effects. Here, we aim to study the effects of different variants on the intestinal epithelium. We present three instances of pediatric inflammatory bowel disease (IBD), displaying varying clinical symptoms and severity levels, and associated with different heterozygous missense mutations in TTC7A. Intestinal organoids derived from patients show dissimilar epithelial phenotypes and exhibit differences in growth, morphology, apicobasal polarity, responses to specific drugs, TTC7A expression, and transcriptional profiles. The findings of our study suggest differences in pathobiology between individuals with different TTC7A mutations. This investigation enhances our comprehension of TTC7A-related conditions and can have implications for developing targeted therapies for TTC7A-associated disorders.

genetics↗

bioRxiv2024

Anticodon Engineered Transfer RNAs (ACE-tRNAs) are a Platform Technology for Suppressing Nonsense Mutations

Nonsense mutations arise from single nucleotide substitutions that result in premature termination codons (PTCs). PTCs result in little to no full-length protein production and loss of mRNA expression through the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate that anticodon engineered (ACE-) tRNAs efficiently suppress the most prevalent cystic fibrosis (CF) causing PTCs, promoting significant rescue of endogenous cystic fibrosis transmembrane conductance regulator (CFTR) transcript abundance and channel function in different model systems. We demonstrate that our best-performing ACE-tRNA, that decodes all UGA PTCs to a leucine amino acid, markedly rescues CFTR channel function from the most prevalent CF causing PTCs that arise from non-leucine encoding codons. Using this single ACE-tRNA variant, we demonstrate significant rescue of CFTR channel function in an immortalized airway cell line and two different primary CF patient-derived intestinal cell models with CFTR nonsense mutations. Thus, ACE-tRNAs have promise as a platform therapeutic for CF and other nonsense-associated diseases.

molecular biology↗

bioRxiv2022

Redefining hypo- and hyper-responding phenotypes of CFTR mutants for understanding and therapy.

Mutations in CFTR cause misfolding and decreased or absent ion-channel function, resulting in the disease Cystic Fibrosis. Fortunately, a triple-modulator combination therapy (Trikafta) has been FDA approved for 178 mutations, including all patients who have F508del on one allele. That so many CFTR mutants respond well to modulators developed for a single mutation is due to the nature of the folding process of this multidomain protein. We have addressed the question What characterizes the exceptions: the mutants that functionally respond either not or extremely well. A functional response is the product of the number of CFTR molecules on the cell surface, open probability, and conductivity of the CFTR chloride channel. By combining biosynthetic radiolabeling with protease-susceptibility assays, we have followed CF-causing mutants during early and late stages of folding in presence and absence of modulators. Most CFTR mutants showed typical biochemical responses for each modulator, such as a TMD1 conformational change or an increase of (cell-surface) stability, regardless of a functional response. These modulators thus should still be considered for hypo-responder genotypes. Understanding both biochemical and functional phenotypes of outlier mutations will boost our insights into CFTR folding and misfolding, and lead to improved therapeutic strategies.

biochemistry↗

bioRxiv2022

High-Throughput Functional Assay in Cystic Fibrosis Patient-Derived Organoids Allows Drug Repurposing

Cystic fibrosis (CF) is a rare hereditary disease caused by mutations in the CFTR gene. Recent therapies enable effective restoration of CFTR function of the most common F508del CFTR mutation. This shifts the unmet clinical need towards people with rare CFTR mutations such as nonsense mutations, of which G542X and W1282X are most prevalent. CFTR function measurements in patient-derived cell-based assays played a critical role in preclinical drug development for CF and may play an important role to identify new drugs for people with rare CFTR mutations. Here, we miniaturized the previously described forskolin induced swelling (FIS) assay in intestinal organoids from a 96-wells to a 384-wells plate screening format. Using this novel assay, we tested CFTR increasing potential of a 1400-compound FDA-approved drug library in organoids from donors with W1282X/W1282X CFTR nonsense mutations. The 384-wells FIS-assay demonstrated uniformity and robustness based on CV and Z-factor calculations. In the primary screen, the top 5 compound combinations that increased CFTR function all contained at least one statin. In the secondary screen, we indeed verified that four out of the five statins, Mevastatin; Lovastatin; Simvastatin and Fluvastatin increased CFTR function when combined with CFTR modulators. Statin-induced CFTR rescue was W1282X specific, as increased CFTR function was not shown for patient-derived organoids harbouring R334W/R334W and F508del/F508del mutations. Future studies should focus on elucidating genotype specificity and mode-of-action of statins into more detail. This study exemplifies proof-of-principle of large-scale compound screening in a functional assay using patient derived organoids. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/500147v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@fccbfdorg.highwire.dtl.DTLVardef@b459ceorg.highwire.dtl.DTLVardef@7414fborg.highwire.dtl.DTLVardef@10d9c35_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Evaluating CRISPR-based Prime Editing for cancer modeling and CFTR repair in intestinal organoids

Prime editing is a recently reported genome editing tool employing a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. The technique holds great promise for clinical application due to its versatility. Here, we explore the use of prime editing in human intestinal organoids. Common TP53 mutations were modeled in human adult stem cell with notable efficiency differences. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9-mediated homology directed repair and adenine base editing on the CFTR-R785* mutation. Despite encountering varying editing efficiencies and undesired mutations, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.

genetics↗