AR Knockout Cell Line (HEK 293)

The potassium channel tetramerization domain containing 13 (KCTD13) protein is a substrate-specific adapter for cullin3-based E3 ubiquitin ligase. Patients with copy number variants at this locus exhibit genitourinary tract anomalies. In this study, we show that decreased androgen receptor (AR) protein level correlated with increased AR ubiquitination in the testis of Kctd13-deficient mice, suggesting that KCTD13 inhibits AR ubiquitination. KCTD13 increased CUL3-dependent AR ubiquitination but had no effect on CUL3 binding to AR, confirming the role of KCTD13 as an adaptor of CUL3 ligase. Recombinant KCTD13 directly binds to recombinant AR, and the BTB domain of KCTD13 is critical for binding both the N-terminal domain of AR and STUB1. Moreover, KCTD13 dose-dependently decreased STUB1 binding to AR resulting in decreased AR ubiquitination. KCTD13 ΔBTB was unable to bind to AR and subsequently failed to block STUB1-mediated AR ubiquitination, strongly suggesting that reduced AR ubiquitination is dependent on KCTD13 ability to dissociate AR/STUB1 complex. Furthermore, KCTD13 increased the expression of AR target gene, FOXJ1, whereas KCTD13 ΔBTB had no effect. Our data reveal a distinctive mode of action of KCTD13 on AR ubiquitination depending on the E3 ubiquitin ligase involved: (1) KCTD13 increased CUL3-dependent AR ubiquitination but had no effect on CUL3 binding to AR; and (2) KCTD13 decreased STUB1-mediated AR ubiquitination by decreasing STUB1 binding to AR thus preventing AR ubiquitination. We hypothesize that in the testes of Kctd13-deficient mice, the absence of KCTD13 results in increased binding of STUB1 to AR leading to increased AR ubiquitination and degradation.

: GM3 Synthase Deficiency (GM3SD) is a rare autosomal recessive neurodevelopmental disease characterized by recurrent seizures and neurological deficits. The disorder stems from mutations in the gene, encoding GM3 synthase (GM3S), a key enzyme in ganglioside biosynthesis. While enzyme deficiencies affecting ganglioside catabolism are well-documented, the consequences of impaired ganglioside biosynthesis remain less explored. : To investigate GM3SD, we used a Human Embryonic Kidney 293-T (HEK293-T) knockout (KO) cell model generated via CRISPR/Cas9 technology. Lipid composition was assessed via high-performance thin-layer chromatography (HPTLC); glycohydrolase activity in lysosomal and plasma membrane (PM) fractions was enzymatically analyzed. Lysosomal homeostasis was evaluated through protein content analysis and immunofluorescence, and cellular bioenergetics was measured using a luminescence-based assay. : Lipidome profiling revealed a significant accumulation of lactosylceramide (LacCer), the substrate of GM3S, along with increased levels of monosialyl-globoside Gb5 (MSGb5), indicating a metabolic shift in glycosphingolipid biosynthesis. Lipid raft analysis revealed elevated cholesterol levels, which may impair microdomain fluidity and signal transduction. Furthermore, altered activity of lysosomal and plasma membrane (PM)-associated glycohydrolases suggests secondary deregulation of glycosphingolipid metabolism, potentially contributing to abnormal lipid patterns. In addition, we observed increased lysosomal mass, indicating potential lysosomal homeostasis dysregulation. Finally, decreased adenosine triphosphate (ATP) levels point to impaired cellular bioenergetics, emphasizing the metabolic consequences of GM3SD. : Together, these findings provide novel insights into the molecular alterations associated with GM3SD and establish the HEK293-T KO model as a promising platform for evaluating potential therapeutic strategies.

OBJECTIVE:Micropenis is a condition with significant physical and psychological implications caused mainly by decreased androgen action in penile development. Kctd13-knockout (Kctd13-KO) mice have micropenis, cryptorchidism, and fertility defects because of reduced levels of androgen receptor (AR) and SOX9. We hypothesized that normalizing the levels of AR and SOX9 in the Kctd13-KO penis could help us to understand the mechanism of action of these signaling pathways on penile development. METHODS:We generated transgenic mice lacking Kctd13 and conditionally expressing AR in the urethral mesenchyme after Cre activation with Twist2 (Kctd13-KO; AR-CMV; Twist2; herein called AR+), and Sox9 in the urethral epithelium after Cre activation with Shh (Kctd13-KO; Sox9-CAG; Shh; herein called SOX9+). Mice penile morphology, fertility, and the effect of KCTD13 on AR and SOX9 ubiquitination were evaluated. RESULTS AND DISCUSSION:Kctd13-KO micropenis phenotype was rescued after increasing levels of penile AR or SOX9 as transgenic AR+ and SOX9+ mice have longer penile lengths than Kctd13-KO mice and are comparable to WT mice. In addition, male-urogenital-mating-protuberance and the baculum were significantly shorter and narrower in Kctd13-KO mice compared with transgenic AR+ and SOX9+ mice. The position of the urethral meatus was similar and orthotopic in location in Kctd13-KO, AR+, SOX9+, and WT penises indicating that none of these mice had hypospadias. The subfertility of AR+ and SOX9+ mice was improved. The ectopic expression of KCTD13 in HEK293 cells strongly reduced AR ubiquitination which is abolished when the proteasome pathway is inhibited and this process is mediated by the ubiquitin ligase, STUB1. The effect of KCTD13 on SOX9 ubiquitination is minimal. CONCLUSION:KCTD13 regulates AR ubiquitination by modulating STUB1 binding to AR. Penile restoration of AR and SOX9 improved penile development in Kctd13-KO mice allowing us to discern the contribution from individual signaling pathways and cell types in penile development.