Biology subjects

Ebert, L. K.

Publications and source records attributed to Ebert, L. K..

5 recordsLinked to original sources

bioRxiv2026

INPP5E interactome reveals novel connections to growth factor signaling

Primary cilia are sensory cell membrane protrusions whose malfunction causes diseases known as ciliopathies. Joubert syndrome (JBTS) is a rare recessive ciliopathy causing brain malformations and kidney cysts, among other manifestations. A key player in JBTS is INPP5E, a ciliary phosphoinositide lipid phosphatase. Although INPP5E regulates ciliary growth factor signaling, the molecular mechanisms remain poorly understood. Herein, we show that a constitutively active growth factor receptor (PDGFR-D842V) stimulates INPP5E tyrosine phosphorylation, as does the SRC tyrosine kinase. INPP5E tyrosine phosphorylation did not affect its enzyme activity but was associated with stronger binding to SH3GL1, an endocytic regulator, and SNX9, a phosphoinositide-binding protein involved, like INPP5E, in growth factor-induced ciliary ectovesicle release. Our INPP5E interactomic studies identified other growth factor signaling regulators, including among others: SIN1 (the phosphoinositide-binding subunit of the mTORC2 complex), STRAP (a TGF{beta} and PI3K/AKT signaling regulator), GRB2 (a growth factor receptor adaptor) and the JBTS-associated proteins AHI1 and NPHP1. Moreover, INPP5E strongly interacted with the phosphopeptide-binding 14-3-3 proteins, and did so only in presence of serine-85, a phosphorylated INPP5E residue. Lastly, we found that INPP5E differentially regulates two cilium-dependent growth factor signaling pathways in fibroblasts. Thus, while INPP5E downregulated PDGF-induced AKT phosphorylations, it upregulated TGF{beta}-induced SMAD2 and ERK phosphorylations. Altogether, our work provides important clues on the cilia-dependent actions of growth factors, with implications for ciliopathies like JBTS.

biochemistry↗

bioRxiv2024

The nephronophthisis protein GLIS2/NPHP7 is required for the DNA damage response in kidney tubular epithelial cells

Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease representing the most frequent genetic cause of end-stage kidney failure in children and adolescents. NPH is caused by genetic variants in >20 NPHP genes. While nearly all NPHP genes encode ciliary proteins, classifying NPH as a renal ciliopathy, there is evidence for a pathogenic role of a compromised DNA damage response (DDR). Here, we present a novel Nphp7/Glis2-deficient mouse model with an early stop codon using CRISPR/Cas9-mediated genome editing (Glis2Y122X). Homozygous mice develop cystic kidney disease with significant fibrosis at a higher age. Interestingly, the kidneys of these animals exhibit an accumulation of DNA damage (DD) early on, even before any functional impairment of the kidneys becomes apparent. Interactome analysis for GLIS2 revealed an array of DDR-related proteins within the GLIS2 protein complex. Consistent with the in vivo data, the knockdown of Glis2 in kidney epithelial cells led to increased DNA damage. Moreover, supporting the role of GLIS2 in the DDR, we demonstrate that a substantial proportion of GLIS2 is present within the chromatin fraction of cells which is further increased upon UV-induced DD. Live-cell imaging revealed the rapid recruitment of GFP-tagged GLIS2 to sites of laser-induced DD, a response diminished in Glis2Y122X and a variant of Glis2 resembling a known patient mutation. Overall, our data provide compelling evidence for the direct involvement of GLIS2 in the DNA damage response, highlighting the loss of genome stability as an important factor contributing to the pathogenesis of renal ciliopathies. Author SummaryNephronophthisis (NPH) is a rare inherited kidney disease characterized by cyst formation, fibrosis, and kidney failure at a young age. It is typically caused by mutations in genes essential for the proper function of cilia, small sensory, antenna-like cell protrusions. However, emerging evidence suggests that genome instability and impaired DNA repair may also contribute to NPH. Thus, in some cases, ciliopathies may result from defects in nuclear proteins rather than ciliary proteins. To investigate this, we generated a mouse model with a defective NPHP7/Glis2 gene using genome-editing tools. The encoded protein GLIS2 has been described to be in the cilium and the nucleus, while its detailed function remained unclear. As these mice aged, they exhibited signs of DNA damage and later developed cystofibrotic kidney disease. Consistently, we demonstrated that the GLIS2 protein interacts with key DNA repair proteins and is rapidly recruited to sites of DNA damage and repair. Thus, inefficient DNA damage repair appears to contribute to kidney disease in this mouse model. These findings underscore the crucial role of genome stability in preventing kidney disease, providing new insights into the underlying causes of NPH.

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↗

bioRxiv2021

JADE family proteins regulate proteasome abundance and activity

The JADE protein family (JADE1/2/3) has been implicated in a broad range of cellular functions, including WNT signaling, cell cycle control, regulated cell death, and transcriptional regulation through histone acetyltransferase complexes. All three paralogs share high sequence similarity and contain two PHD zinc finger domains. JADE1 has additionally been associated with cilia-related proteins and genetic disorders affecting kidney architecture and function. Despite their widespread expression, the molecular roles of JADE proteins remain incompletely understood. Here, we identify JADE proteins as regulators of proteasome abundance and activity. Using kidney cells as a model, we demonstrate that loss of any single JADE protein led to a marked upregulation of almost all components of the 26S proteasome. Regulation occurred at the post-translational level and was not the consequence of increased transcription. Consistent with a role for JADE proteins in regulating overall proteasomal abundance, JADE-deficient cells displayed elevated proteasomal activity, while ectopic expression of JADE1, JADE2, or JADE3 reduced proteasomal function. Co-immunoprecipitation experiments confirmed the interaction between JADE1 and multiple proteasomal subunits, supporting a direct role of JADE proteins in modulating proteasome turnover, stability and abundance. These findings reveal a novel function of JADE proteins in proteostasis and suggest that their previously reported cellular roles may, in part, be mediated through regulation of the ubiquitin-proteasome system.

cell biology↗

bioRxiv2018

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6N mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

genetics↗