Ubiquitin Specific Protease USP48 Destabilizes NF-κB/p65 in Retinal Pigment Epithelium Cells.

Activation of NF-κB transcription factor is strictly regulated to accurately direct cellular processes including inflammation, immunity, and cell survival. In the retina, the modulation of the NF-κB pathway is essential to prevent excessive inflammatory responses, which plays a pivotal role in many retinal neurodegenerative diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and inherited retinal dystrophies (IRDs). A critical cytokine mediating inflammatory responses in retinal cells is tumor necrosis factor-alpha (TNFα), leading to the activation of several transductional pathways, including NF-κB. However, the multiple factors orchestrating the appropriate regulation of NF-κB in retinal cells still remain unclear. The present study explores how the ubiquitin-specific protease 48 (USP48) downregulation impacts the stability and transcriptional activity of NF-κB/p65 in retinal pigment epithelium (RPE), at both basal conditions and following TNFα stimulation. We described that USP48 downregulation stabilizes p65. Notably, the accumulation of p65 is mainly detectable in the nuclear compartment and it is accompanied by an increased NF-κB transcriptional activity. These results delineate a novel role of USP48 in negatively regulating NF-κB in retinal cells, providing new opportunities for therapeutic intervention in retinal pathologies.

Human Genetic Diseases Linked to the Absence of NEMO: An Obligatory Somatic Mosaic Disorder in Male.

De novo somatic mutations are well documented in diseases such as neoplasia but are rarely reported in rare diseases. Hovewer, severe genetic diseases that are not compatible with embryonic development are caused exclusively by deleterious mutations that could only be found as mosaic and not as inherited mutations. We will review here the paradigmatic case of Incontinentia Pigmenti, a rare X-linked dominant disease caused by deficiency of the NEMO (also called IKKgamma) protein, which plays a pivotal role in tissue homeostasis. The loss-of-function mutations of NEMO are embryonically lethal in males while females survive because of unbalanced X-inactivation due to NEMO wild type (WT) expressing cells survival despite of NEMO mutant expressing cells. The few surviving IP males are obligatory mosaic mutants with the typical clinical presentation of IP in female. Indeed, the IP pathogenesis in the female and most likely also in the male somatic mosaics is based on the cellular effects of an impaired NEMO activity, but in the context of the interaction of genetically different cells in the affected tissue, which might underline the inflammatory status.

Methods to Study the Effect of IKK Inhibition on TNF-Inducing Apoptosis and Necroptosis in Cultured Cells.

The engagement of TNF on TNFR can result in cell survival or cell death depending on the different complex formation downstream this interaction. Here we describe reagents and assay procedures that can be used to study caspase-independent cell death (necroptosis) in cultured cells, in response to pharmacological interventions with NF-kappaB and death inhibitors. We provide protocol to detect death-specific proteins using immunoblot and to dissect necrosome complex by sequential co-immunoprecipitation of death-specific components during necroptosis.