Biology subjects

May-Simera, H. L.

Publications and source records attributed to May-Simera, H. L..

5 recordsLinked to original sources

bioRxiv2025

Maturation-dependent complement production and C3 processing in human retinal pigment epithelium cells

Induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells, closely resembling healthy RPE, offer valuable insights for retinal disease modelling. This study evaluates immature and mature phenotypes of iPSC-RPE and ARPE-19 cells, comparing RPE characteristics, cell-associated complement profiles, and TGF-{beta}1-mediated stress responses across transcript expression, secretion, and protein levels. Statistical analyses were performed using independent means t-test and Wilcoxon rank-sum test. Mature iPSC-RPE cells exhibited characteristic RPE-morphology, with elevated secretion of complement components (C3, FH/FHL-1, FI), unique FB secretion, and apical complement localisation. Intracellular C3 processing revealed cleavage products different from known blood-derived C3 fragments and showed maturation-dependent differences; only mature iPSC-RPE cells secreted active C3 forms (C3(H2O), C3a) and exhibited the intact C3 {beta}-chain. Mature ARPE-19 and iPSC-RPE cells demonstrated resistance to TGF-{beta}1 treatment, which reduced complement secretion without affecting C3a release. In conclusion, ARPE-19 and iPSC-RPE cells demonstrated local production of complement components and maturation-dependent intracellular processing of C3 into active forms. These findings highlight the impact of RPE maturation on local C3 activation, providing a basis for future studies on C3 functionality in the RPE and its potential pathological effects. Impact statementCell state-dependent local processing of complement C3 in retinal pigment epithelium cells generates active C3 forms that may evade anti-C3 therapies, influencing age-related macular degeneration and treatment outcomes. Funding statementThis project has received funding from the PRO RETINA, Amberg, Germany, under grant agreement number "Pro-Re/Projekt/Schikora - Pauly.05-2022", and Deutsche Forschungsgemeinschaft (DFG), under grant agreement number "498244102 (MA 6139/5-1)". Open access funding provided by the Open Access Publishing Fund of University Marburg. Funding sources were not involved in study design, data collection and interpretation, or the decision to submit the work for publication. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/636432v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@6c63b3org.highwire.dtl.DTLVardef@58010org.highwire.dtl.DTLVardef@11ab625org.highwire.dtl.DTLVardef@ac4924_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

The BBS/CCT chaperonin complex ensures the localization of the adhesion G protein-coupled receptor ADGRV1 to primary cilia

Primary cilia are antenna-like sensory organelles present on almost all eukaryotic cells. Their sensory capacity relies on receptors, in particular G-protein-coupled receptors (GPCRs) which localize to the ciliary membrane. Here we show that ADGRV1, a member of the GPCR subfamily of adhesion GPCRs, is part of a large protein network, interacting with numerous proteins of a comprehensive ciliary proteome. ADGRV1 is localized to the base of prototypic primary cilia in cultured cells and the modified primary cilia of retinal photoreceptors, where it interacts with TRiC/CCT chaperonins and the Bardet Biedl syndrome (BBS) chaperonin-like proteins. Knockdown of ADGRV1, CCT2 and 3, and BBS6 result in common ciliogenesis phenotypes, namely reduced ciliated cells combined with shorter primary cilia. In addition, the localization of ADGRV1 to primary cilia depends on the activity of a co-complex of TRiC/CCT chaperonins and the BBS chaperonin-like proteins. In the absence of components of the TRiC/CCT-BBS chaperonin co-complex, ADGRV1 is depleted from the base of the primary cilium and degraded via the proteasome. Defects in the TRiC/CCT-BBS chaperonin may lead to an overload of proteasomal degradation processes and imbalanced proteostasis. Dysfunction or absence of ADGRV1 from primary cilia may underly the pathophysiology of human Usher syndrome type 2 and epilepsy caused by mutations in ADGRV1.

cell biology↗

bioRxiv2024

Loss of KIF13B causes time-dependent changes in ciliary polycystin-2 levels and extracellular vesicle release

Dynamic control of ciliary membrane protein content is crucial for the organelles homeostasis and signaling function and involves removal of ciliary components by BBSome-mediated export, endocytic retrieval and/or extracellular vesicle (EV) shedding. We report that KIF13B regulates ciliary protein composition and EV shedding in cultured kidney epithelial cells, with effects that vary over time. In early stages of ciliation Kif13b-/- cells aberrantly accumulate PC2, FLOT1, and HGS within cilia. These cells also produce fewer small EVs through the GW4869-sensitive, nSMase2 pathway, and release large EVs enriched with CCDC198 and the centriole distal appendage protein CCDC92, which also localizes to the ciliary tip. Upon cilia maturation, Kif13b-/- cells accelerate large EV release of numerous ciliary proteins, including PC2, BBSome components, and IFT proteins, which correlates with gradual depletion of CCDC92 and PC2 from the ciliary tip and shaft, respectively. Furthermore, over time, Kif13b-/- cells show an upregulation in the release of small EVs, which differ in composition from wild-type small EVs. Specifically, the mutant small EVs lack several proteins that are enriched in small EVs from BBSome-deficient cells, such as the palmitoyl transferase ZDHHC5, which localizes to cilia, accumulates within cilia of BBSome-deficient cells, and regulates ciliary length and PC2 levels. Collectively, our work suggests that KIF13B acts at the level of centriole distal appendages to limit ciliary protein entrance and promote endocytic retrieval downstream of the BBSome. Furthermore, this study shows for the first time that CCDC198 and ZDHHC5 localize to primary cilia, suggesting they are potential novel ciliopathy candidates.

cell biology↗

bioRxiv2024

Loss of Bbs8 leads to cystic kidney disease in mice and affects tubulin acetylation through HDAC2

Primary cilia dysfunction underlies a group of severe disorders known as ciliopathies. These include Bardet-Biedl syndrome (BBS), which is caused by mutations in BBS genes encoding for components of ciliary protein complexes essential for the assembly and maintenance of primary cilia. As in most ciliopathies, a hallmark feature of BBS is the development of cystic kidney disease. However, the molecular mechanisms linking ciliary dysfunction to cystogenesis remain incompletely understood. Here, we show that Bbs8-/- mice develop late-onset cystic kidney disease accompanied by increased regulated cell death and fibrosis. While the number and length of cilia are not affected, loss of BBS8 reduces K40 acetylation of -tubulin within primary cilia, compromising ciliary stability. Notably, proteomic analysis revealed a significant upregulation of histone deacetylase HDAC2 in Bbs8-/- kidneys, which we confirmed in Bbs8-/- mouse embryonic fibroblasts (MEFs) and in urine-derived renal epithelial cells (URECs) from a BBS8 patient. We further demonstrate the protein interaction between BBS8 and HDAC2, implicating a disrupted BBS8-HDAC2 regulatory axis in disease pathogenesis. Consistent with a role of excessive HDAC2 activity in the BBS8 deficient cells, pharmacological inhibition of HDAC2 restored tubulin acetylation in BBS8 urine-derived cells. Thus, modulation of HDAC2 activity may represent a strategy to alter ciliary stability in vivo which could explain positive effects of class I specific HDAC inhibitors in models of cystic kidney disease.

genetics↗

bioRxiv2023

Analysis of human BBS protein homologues in insects support alternative non-ciliary functions

Cilia and flagella were one of the characteristic traits of the last eukaryotic common ancestor and as such, are highly conserved among eukaryotes. Their proteomic makeup is consequently remarkably similar throughout all eukaryotic lineages. Recently, one subgroup of ciliary transport proteins in mammalian cells, the Bardet-Biedl Syndrome (BBS) proteins, was shown to have the ability to traverse the nuclear envelope, and to engage in protein-protein-interactions that modulate gene expression, signalling cascades, and cell homeostasis. Insects have been critically understudied in cilia biology because of their highly specialised cilia being localised on only a small subset of cell types. In this study, we present evidence that the BBSome, a hetero-octameric ciliary transport complex of BBS proteins, is largely conserved in multiple insect lineages. Using the honeybee Apis mellifera as a study system to explore BBS-associated gene expression, our analyses suggest that not all BBSome-associated genes are expressed equally, indicating possible non-ciliary functions. We also demonstrate that the expression of individual BBS proteins varies significantly between the tissues of queens and males in A. mellifera, especially in neuronal tissue. This result raises the question of what role BBS proteins play in these tissues and whether they are involved in gene regulation in insects. The potential gene regulatory function of BBS proteins should be explored in other eukaryotes due to their high degree of conservation.

evolutionary biology↗