Biology subjects

Prasai, A.

Publications and source records attributed to Prasai, A..

6 recordsLinked to original sources

bioRxiv2026

Bardet-Biedl Syndrome 1 Mutations Differentially Impact BBSome Integrity and its Function in Ciliary Trafficking

Bardet-Biedl Syndrome (BBS) is a pleiotropic ciliopathy marked by retinal degeneration, obesity, polydactyly, renal and reproductive anomalies, and cognitive impairment. BBS1, the most frequently mutated gene in BBS, encodes a key component of the BBSome complex, which is essential for ciliary membrane trafficking. Although BBS1 is known to be essential for proper BBSome function, the effects of disease-associated BBS1 variants on its activity remain incompletely understood. In this study, we examined how patient-derived BBS1 mutations affect BBSome integrity and its role in cargo transport within primary cilia. Our results show that particular BBS1 mutations interfere with distinct stages of BBSome assembly and trafficking. While M390R disrupts initial pre-BBSome assembly at pericentriolar satellites, E224K impairs both the maturation of the pre-BBSome into the BBSome and its movement from pericentriolar satellites to the cilium. In contrast, the R160Q variant preserves BBSome assembly and permits its localization to cilia. It specifically weakens the BBSome-GPCR interaction mediated by TOM1L2, resulting in defective GPR161 export and increased ciliary IFT turnover. Overall, our study establishes a mechanistic framework linking specific BBS1 mutations to distinct defects in BBSome assembly and function. This framework defines functional classes of BBS1 variants and provides deeper insight into the molecular mechanism and severity of Bardet-Biedl Syndrome.

cell biology↗

bioRxiv2025

iAPEX: Improved APEX-based proximity labeling for subcellular proteomics using an enzymatic reaction cascade

Ascorbate peroxidase (APEX) is a versatile labeling enzyme used for live-cell proteomics at high spatial and temporal resolution. However, toxicity of its substrate hydrogen peroxide and background labeling by endogenous peroxidases limit its use to in vitro studies of specific cell types. By combining APEX2 with a D-amino acid oxidase to locally produce hydrogen peroxide, we establish a more versatile, improved APEX (iAPEX) workflow that minimizes hydrogen peroxide toxicity and reduces non-specific background labeling. We employ iAPEX to perform live-cell proteomics of a cellular microdomain, the primary cilium, in previously inaccessible cell lines, leading to the identification of new ciliary proteins. Our study robustly validates common ciliary proteins across two distinct cell lines, while observed differences may reflect heterogeneity in primary cilia proteomes. iAPEX proximity labeling in Xenopus laevis provides a proof-of-concept for future in vivo applications.

biochemistry↗

bioRxiv2023

De-suppression of mesenchymal cell identities and variable phenotypic outcomes associated with knockout of Bbs1.

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue specific, and gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane trafficking and intraflagellar transport (IFT) adaptor protein complex formed by 8 BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically-defined clones. Our results suggest that BBS1 is required for suppression of mesenchymal cell identities as IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, and BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively this work suggests that the dynamic stability of the BBSome is essential for suppression of mesenchymal cell identities as epithelial cells differentiate.

genetics↗

bioRxiv2023

BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed as compensatory cellular process for defective cargo retrieval, the underlying molecular mechanism is poorly understood, particularly in the context of BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Furthermore, activation of the Sonic Hedgehog pathway further enhances CDC42 activity in BBSome-deficient cilia, but not in healthy cells. Inhibition of CDC42 decreased the frequency and duration of ciliary actin polymerization events and lead to the accumulation of the GPR161 receptor in bulges along the axoneme in BBSome-deficient cells during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Moreover, the hyperactivated ciliary CDC42-actin-ectocytosis axis in BBSome-deficient cells results in cilia shortening and loss of ciliary material, potentially impacting disease severity.

cell biology↗

bioRxiv2020

The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells

Bardet-Biedl Syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. Most BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, BBSome, which mediates the trafficking of ciliary cargoes. Although, the structure of the BBSome has been resolved recently, the mechanism of assembly of this complicated complex in living cells is poorly understood. We generated a large library of human retinal epithelial cell lines deficient in particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We performed a comprehensive analysis of these cell lines using biochemical and microscopy approaches. Our data revealed that the BBSome formation is a sequential process including a step of the pre-BBSome assembly at pericentriolar satellites nucleated by BBS4, followed by the translocation of the BBSome into the ciliary base mediated by BBS1.

cell biology↗

bioRxiv2020

Altered hematopoietic system and self-tolerance in Bardet-Biedl Syndrome

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by dysfunction of primary cilia. The immune system of patients with BBS or another ciliopathy has not been investigated, most likely because hematopoietic cells do not form cilia. However, there are multiple indications that the impairment of the processes typically associated with cilia might influence the hematopoietic compartment and immunity. In this study, we analyzed clinical data of BBS patients as well as a corresponding mouse model of BBS4 deficiency. We uncovered that BBS patients have higher incidence of certain autoimmune diseases. BBS patients and animal models have elevated white blood cell levels and altered red blood cell and platelet compartments. Moreover, we observed that BBS4 deficiency alters the development and homeostasis of B cells in mice. Some of the hematopoietic system alterations were caused by the BBS-induced obesity. Overall, our study reveals a connection between a ciliopathy and the alterations of the immune system and the hematopoietic compartment.

genetics↗