Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.     

Lactate promotes macrophage HMGB1 lactylation,acetylation, and exosomal release in polymicrobial sepsis

High circulating levels of lactate and high mobility group box-1 (HMGB1) are associated with the severity and mortality of sepsis. However, it is unclear whether lactate could promote HMGB1 release during sepsis. The present study demonstrated a novel role of lactate in HMGB1 lactylation and acetylation in macrophages during polymicrobial sepsis. We found that macrophages can uptake extracellular lactate via monocarboxylate transporters (MCTs) to promote HMGB1 lactylation via a p300/CBP-dependent mechanism. We also observed that lactate stimulates HMGB1 acetylation by Hippo/YAP-mediated suppression of deacetylase SIRT1 and β-arrestin2-mediated recruitment of acetylases p300/CBP to the nucleus via G protein-coupled receptor 81 (GPR81). The lactylated/acetylated HMGB1 is released from macrophages via exosome secretion which increases endothelium permeability. In vivo reduction of lactate production and/or inhibition of GPR81-mediated signaling decreases circulating exosomal HMGB1 levels and improves survival outcome in polymicrobial sepsis. Our results provide the basis for targeting lactate/lactate-associated signaling to combat sepsis.Subject terms: Infectious diseases, Signal transduction, Epigenetics     

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110–CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110–CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.     

Cdc42 is a substrate for caspases and influences Fas-induced apoptosis

Fas-mediated apoptosis results in the activation of caspases, which subsequently cleave cellular substrates that are essential for normal cell viability. In the present study, we show that the Ras-related GTP-binding protein Cdc42 is susceptible to caspase-catalyzed proteolysis in a number of cell lines, including NIH3T3 fibroblasts, human breast cancer cells (e.g. T47D), and COS-7 cells. Both caspase-3 and caspase-7 were able to catalyze the cleavage of Cdc42, whereas caspase-6 and caspase-8 were without effect. The susceptibility to the caspase-stimulated degradation is specific; although Rac can also serve as a caspase substrate, neither Rho nor Ras is degraded. Caspase sensitivity is conferred by a consensus sequence (DXXD) that lies immediately upstream of the Rho insert regions (residues 122-134) of Cdc42 and Rac. The removal of a stretch of residues (120) that includes the insert region or site-directed mutagenesis of either aspartic acid 118 or 121 within a constitutively active background (i.e. Cdc42(F28L)) as well as a wild-type Cdc42 background yields Cdc42 molecules that provide a marked protection against Fas ligand-induced apoptosis. Overall, these results are consistent with a model in which Cdc42 acts downstream of Fas, perhaps to influence the rate of apoptosis, with the ultimate caspase-mediated degradation of Cdc42 then allowing for a maximal apoptotic response.     

Structural and kinetic analysis of the chemical rescue of the proton transfer function of carbonic anhydrase II

Histidine 64 in human carbonic anhydrase II (HCA II) functions in the catalytic pathway of CO(2) hydration as a shuttle to transfer protons between the zinc-bound water and bulk water. Catalysis of the exchange of (18)O between CO(2) and water, measured by mass spectrometry, is dependent on this proton transfer and was decreased more than 10-fold for H64A HCA II compared with wild-type HCA II. The loss of catalytic activity of H64A HCA II could be rescued by 4-methylimidazole (4-MI), an exogenous proton donor, in a saturable process with a maximum activity of 40% of wild-type HCA II. The crystal structure of the rescued complex at 1.6 A resolution shows 4-MI bound in the active-site cavity of H64A HCA II, through pi stacking interactions with Trp 5 and H-bonding interactions with water molecules. In this location, 4-MI is about 12 A from the zinc and approximates the observed "out" position of His 64 in the structure of the wild-type enzyme. 4-MI appears to compensate for the absence of His 64 and rescues the catalytic activity of the H64A HCA II mutant. This result strongly suggests that the out conformation of His 64 is effective in the transfer of protons between the zinc-bound solvent molecule and solution.     

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.     

Immunolocalization and possible effect of a moth allatotropin-like substance in a fly, Phormia regina (Diptera: Calliphoridae)

Insect allatotropin upregulates the biosynthesis of juvenile hormones by the corpus allatum. We raised two rabbit antisera against the allatotropin of Manduca sexta (Mas AT) using a synthetic, multiple-antigenic-peptide that contains a branching heptalysine core and eight Mas AT molecules. Both antisera recognized specifically the same neurons in the larval brain, frontal ganglion and terminal abdominal ganglion of M. sexta as previously reported by others. Immunoassay showed reactivity specific to the Mas AT. Very low or nearly no cross-reactivity was found for two Mas AT-like peptides, a myotropin from Locusta migratoria and a Mas AT-like peptide deduced from the DNA sequence of Aedes aegypti, respectively. Immunopositive neurons also were identified in adult Phormia regina, Dacus dorsalis, Oncopeltus fasciatus, and Mythimna loreyi, and in larval M. loreyi, Bombyx mori, and Andraca bipunctata. At 20 pmol per 25 μl incubation medium (i.e. 8x10(-7) M), synthetic Mas AT significantly stimulated in vitro juvenile hormone biosynthesis by the corpus allatum of adult, sugar-fed females of P. regina to 2.64-fold that of controls. Thus, this study provides the first demonstration that at the higher end of the physiological concentration range, the Mas AT has allatotropic effect in vitro to CA of non-lepidopterans. However, in vivo functions of Mas AT and/or Mas AT-like peptide in P. regina remain to be defined.