Zinc and zinc transporters in macrophages and their roles in efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis

po 077 identification of dhx30 as an inhibitor of the translation of pro apoptotic mrnas after p53 activation by nutlin

Introduction The transcription factor p53 can be efficiently activated by the small molecule Nutlin-3 without inducing genotoxic stress. Treatment of different cell lines with this small molecule can result in different phenotypes, ranging from cell cycle arrest to apoptosis. HCT116 (colon cancer-derived cells) and SJSA1 (osteosarcoma-derived cells) were used to model the opposite behaviour respectively, by analysing the transcriptional and translational responses after Nutlin-3 treatment. Material and methods Total and polysomal-bound mRNAs were collected and sequenced after 12 hour of 10 uM Nutlin-3 treatment. A bioinformatics analysis of the polysome-enriched mRNAs using Weeder allowed the identification of a 3'UTR motif ('CG-rich') which is enriched in the translationally upregulated genes of SJSA1. The effect of the motif on translation was evaluated after cloning its consensus sequence in the 3'UTR of the b-globin gene, which was put downstream the luciferase reporter. The activity of the construct was evaluated after 12 or 24 hours of Nutlin-3. The same consensus was used for a pull-down experiment followed by mass spectrometry to identify proteins interacting with it. Results and discussions RNA-seq data indicate that HCT116 and SJSA1, although sharing almost completely the transcriptional program lead by p53, show almost no overlap at a translation level. SJSA1 present different pro-apoptotic translationally-upregulated genes after Nutlin-3, which have one or more instances of a CG-rich motif in the 3'UTR. The impact of the motif is to enhance the activity of the luciferase reported when cloned in two copies flanking the 3'UTR of the b-globin gene, but only in SJSA1. A pull-down experiment using an RNA bait with the consensus motif was used to identify interactors, among which DHX30 was deeply studied. DHX30 silencing in HCT116 causes: 1) enhanced the activity of the reporter construct after Nutlin; 2) polysomal association of selected mRNAs containing the motif; 3) induction of apoptosis as assessed by Annexin-V staining. In addition, silencing of DHX30 in U2OS cells decreased their survival after Nutlin-3 treatment. Conclusion We show how a p53-dependent transcriptional program can be shaped at a translational level thanks to the action of a CG-rich motif which is enriched in the 3'UTR of some pro-apoptotic mRNAs and that can be bound by DHX30. This protein acts as a translational repressor of mRNAs containing the motif. The exact mechanism and the generalisation of the model are currently being investigated

Atomic structures of TDP-43 LCD segments and insights into reversible or pathogenic aggregation.

The normally soluble TAR DNA-binding protein 43 (TDP-43) is found aggregated both in reversible stress granules and in irreversible pathogenic amyloid. In TDP-43, the low-complexity domain (LCD) is believed to be involved in both types of aggregation. To uncover the structural origins of these two modes of β-sheet-rich aggregation, we have determined ten structures of segments of the LCD of human TDP-43. Six of these segments form steric zippers characteristic of the spines of pathogenic amyloid fibrils; four others form LARKS, the labile amyloid-like interactions characteristic of protein hydrogels and proteins found in membraneless organelles, including stress granules. Supporting a hypothetical pathway from reversible to irreversible amyloid aggregation, we found that familial ALS variants of TDP-43 convert LARKS to irreversible aggregates. Our structures suggest how TDP-43 adopts both reversible and irreversible β-sheet aggregates and the role of mutation in the possible transition of reversible to irreversible pathogenic aggregation

Caregiver Recognition of Childhood Diarrhea, Care Seeking Behaviors and Home Treatment Practices in Rural Burkina Faso: A Cross-Sectional Survey

Introduction: To design effective national diarrhea control programs, including oral rehydration solution (ORS) and therapeutic zinc supplementation, information is needed on local perceptions of illness, external care seeking behaviors, and home treatment practices. Methods: A cross-sectional, community-based household survey was conducted in the Orodara Health District, Burkina Faso. Caregivers of 10,490 children,27 months were interviewed to assess child diarrhea prevalence and related car

Nf2/Merlin controls spinal cord neural progenitor function in a Rac1/ErbB2-dependent manner

Objective: Individuals with the neurofibromatosis type 2 (NF2) cancer predisposition syndrome develop spinal cord glial tumors (ependymomas) that likely originate from neural progenitor cells. Whereas many spinal ependymomas exhibit indolent behavior, the only treatment option for clinically symptomatic tumors is surgery. In this regard, medical therapies are unfortunately lacking due to an incomplete understanding of the critical growth control pathways that govern the function of spinal cord (SC) neural progenitor cells (NPCs). Methods: To identify potential therapeutic targets for these tumors, we leveraged primary mouse Nf2-deficient spinal cord neural progenitor cells. Results: We demonstrate that the Nf2 protein, merlin, negatively regulates spinal neural progenitor cell survival and glial differentiation in an ErbB2-dependent manner, and that NF2-associated spinal ependymomas exhibit increased ErbB2 activation. Moreover, we show that Nf2-deficient SC NPC ErbB2 activation results from Rac1-mediated ErbB2 retention at the plasma membrane. Significance: Collectively, these findings establish ErbB2 as a potential rational therapeutic target for NF2-associated spinal ependymoma

Tyrosine Phosphorylation of Rac1: A Role in Regulation of Cell Spreading

Rac1 influences a multiplicity of vital cellular- and tissue-level control functions, making it an important candidate for targeted therapeutics. The activity of the Rho family member Cdc42 has been shown to be modulated by tyrosine phosphorylation at position 64. We therefore investigated consequences of the point mutations Y64F and Y64D in Rac1. Both mutations altered cell spreading from baseline in the settings of wild type, constitutively active, or dominant negative Rac1 expression, and were accompanied by differences in Rac1 targeting to focal adhesions. Rac1-Y64F displayed increased GTP-binding, increased association with βPIX, and reduced binding with RhoGDI as compared with wild type Rac1. Rac1-Y64D had less binding to PAK than Rac1-WT or Rac1-64F. In vitro assays demonstrated that Y64 in Rac1 is a target for FAK and Src. Taken together, these data suggest a mechanism for the regulation of Rac1 activity by non-receptor tyrosine kinases, with consequences for membrane extension

The N-Terminal Domain of the Drosophila Retinoblastoma Protein Rbf1 Interacts with ORC and Associates with Chromatin in an E2F Independent Manner

The retinoblastoma (Rb) tumor suppressor protein can function as a DNA replication inhibitor as well as a transcription factor. Regulation of DNA replication may occur through interaction of Rb with the origin recognition complex (ORC).We characterized the interaction of Drosophila Rb, Rbf1, with ORC. Using expression of proteins in Drosophila S2 cells, we found that an N-terminal Rbf1 fragment (amino acids 1-345) is sufficient for Rbf1 association with ORC but does not bind to dE2F1. We also found that the C-terminal half of Rbf1 (amino acids 345-845) interacts with ORC. We observed that the amino-terminal domain of Rbf1 localizes to chromatin in vivo and associates with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4.Our results suggest that Rbf1 can associate with ORC and chromatin through domains independent of the E2F binding site. We infer that Rbf1 may play a role in regulating replication directly through its association with ORC and/or chromatin factors other than E2F. Our data suggest an important role for retinoblastoma family proteins in cell proliferation and tumor suppression through interaction with the replication initiation machinery