NRF2 and primary cilia: An emerging partnership

When not dividing, many cell types target their centrosome to the plasma membrane, where it nucleates assembly of a primary cilium, an antenna-like signaling structure consisting of nine concentric microtubule pairs surrounded by membrane. Primary cilia play important pathophysiological roles in many tissues, their dysfunction being associated with cancer and ciliopathies, a diverse group of congenital human diseases. Several recent studies have unveiled functional connections between primary cilia and NRF2 (nuclear factor erythroid 2-related factor 2), the master transcription factor orchestrating cytoprotective responses to oxidative and other cellular stresses. These NRF2-cilia relationships are reciprocal: primary cilia, by promoting autophagy, downregulate NRF2 activity. In turn, NRF2 transcriptionally regulates genes involved in ciliogenesis and Hedgehog (Hh) signaling, a cilia-dependent pathway with major roles in embryogenesis, stem cell function and tumorigenesis. Nevertheless, while we found that NRF2 stimulates ciliogenesis and Hh signaling, a more recent study reported that NRF2 negatively affects these processes. Herein, we review the available evidence linking NRF2 to primary cilia, suggest possible explanations to reconcile seemingly contradictory data, and discuss what the emerging interplay between primary cilia and NRF2 may mean for human health and disease.This work was funded by European Regional Development Fund (ERDF/FEDER)-cofunded grants from the Spanish Ministry of Economy and Competitiveness (MINECO) to F.R.G.G. (SAF2015-66568-R and RYC2013-14887). A.M.H. was supported by a FEDER-cofunded predoctoral contract from the Community of Madrid governmen

The Deubiquitinating Enzyme USP48 Interacts with the Retinal Degeneration-Associated Proteins UNC119a and ARL3

Proteins related to the ubiquitin-proteasome system play an important role during the differentiation and ciliogenesis of photoreceptor cells. Mutations in several genes involved in ubiquitination and proteostasis have been identified as causative of inherited retinal dystrophies (IRDs) and ciliopathies. USP48 is a deubiquitinating enzyme whose role in the retina is still unexplored although previous studies indicate its relevance for neurosensory organs. In this work, we describe that a pool of endogenous USP48 localises to the basal body in retinal cells and provide data that supports the function of USP48 in the photoreceptor cilium. We also demonstrate that USP48 interacts with the IRD-associated proteins ARL3 and UNC119a, and stabilise their protein levels using different mechanisms. Our results suggest that USP48 may act in the regulation/stabilisation of key ciliary proteins for photoreceptor function, in the modulation of intracellular protein transport, and in ciliary trafficking to the photoreceptor outer segment

EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO

Ellis van Creveld syndrome and Weyers acrofacial dysostosis are two rare genetic diseases affecting skeletal development. They are both ciliopathies, as they are due to malfunction of primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae and are required for Hedgehog signaling, a key pathway during skeletal morphogenesis. These ciliopathies are caused by mutations affecting the EVC-EVC2 complex, a transmembrane protein heterodimer that regulates Hedgehog signaling from inside primary cilia. Despite the importance of this complex, the mechanisms underlying its stability, targeting and function are poorly understood. To address this, we characterized the endogenous EVC protein interactome in control and Evc-null cells. This proteomic screen confirmed EVC’s main known interactors (EVC2, IQCE, EFCAB7), while revealing new ones, including USP7, a deubiquitinating enzyme involved in Hedgehog signaling. We therefore looked at EVC-EVC2 complex ubiquitination. Such ubiquitination exists but is independent of USP7 (and of USP48, also involved in Hh signaling). We did find, however, that monoubiquitination of EVC-EVC2 cytosolic tails greatly reduces their protein levels. On the other hand, modification of EVC-EVC2 cytosolic tails with the small ubiquitin-related modifier SUMO3 has a different effect, enhancing complex accumulation at the EvC zone, immediately distal to the ciliary transition zone, possibly via increased binding to the EFCAB7-IQCE complex. Lastly, we find that EvC zone targeting of EVC-EVC2 depends on two separate EFCAB7-binding motifs within EVC2’s Weyers-deleted peptide. Only one of these motifs had been characterized previously, so we have mapped the second herein. Altogether, our data shed light on EVC-EVC2 complex regulatory mechanisms, with implications for ciliopathies

Mutations in SCNM1 cause orofaciodigital syndrome due to minor intron splicing defects affecting primary cilia

Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity.This work was supported by a grant from the Spanish Ministry of Science and Innovation (PID2019-105620RB-I00/AEI/10.13039/501100011033)

Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal

BACKGROUND: Although human embryonic stem cells (hESCs) hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect. CONCLUSIONS/SIGNIFICANCE: Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality