Characterization of Microfibrillar-Associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-Binding Protein Involved in Elastic Fiber Formation [Glycobiology and Extracellular Matrices]

November 24th, 2015 by

Microfibrillar-associated protein 4 (MFAP4) is an extracellular glycoprotein found in elastic fibers with not clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin, fibrillin-1 and -2 as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues F241 and S203 in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.
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Hyaluronan rafts on airway epithelial cells [Glycobiology and Extracellular Matrices]

November 24th, 2015 by Abbadi, A., Lauer, M., Swaidani, S., Wang, A., Hascall, V.

Many cells, including murine airway epithelial cells, respond to a variety of inflammatory stimuli by synthesizing leukocyte-adhesive hyaluronan cables that remain attached to their cell surfaces. This study shows that air-liquid interface cultures of murine airway epithelial cells (AECs) also actively synthesize and release a majority of their HA onto their ciliated apical surfaces to form a heavy chain-hyaluronan (HC-HA) matrix in the absence of inflammatory stimuli. These matrices do not resemble the rope-like HA cables, but occur in distinct sheets or rafts, that can capture and embed leukocytes from cell suspensions. The HC-HA modification involves the transfer of heavy chains from the inter-α-inhibitor (IαI) proteoglycan, which has 2 heavy chains (HC1 and HC2) on its chondroitin sulfate (CS) chain. The transesterification transfer of HCs from CS to HA is mediated by tumor-necrosis-factor-induced-gene 6 (TSG-6), which is upregulated in inflammatory reactions. Because the AEC cultures do not have TSG-6 nor serum, the source of IαI, assays for HCs and TSG-6 were done. The results show that AECs synthesize TSG-6 and their own heavy chain donor (pre-IαI) with a single heavy chain 3 (HC3), which are also constitutively expressed by human renal proximal tubular epithelial cells. These leukocyte adhesive HC3-HA structures were also found in the bronchoalveolar lavage (BAL) of naιve mice, and were observed on their apical ciliated surfaces. Thus, these leukocyte-adhesive HA rafts are now identified as HC3-HA complexes that could be part of a host defense mechanism filling some important gaps in our current understanding of murine airway epithelial biology and secretions.

The Tomato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor I-2 Couples DNA-Binding to Nucleotide-Binding Domain Nucleotide Exchange [Plant Biology]

November 24th, 2015 by

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognise and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA≈dsDNA>ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Since DNA binding by I-2 was decreased in the presence of ADP when compared to ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA-binding is a general property of at least a subset of NLR proteins and NLR activation is directly linked to its activity at DNA.
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Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter. [Cell Biology]

November 24th, 2015 by

Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g., Doxorubicin; DOX). This mechanism serves as a "safe-house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and Pgp-substrate, di-2-pyridylketone-4, 4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization (LMP). This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant (MDR) cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor micro-environment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally-related analogue, di-2-pyridylketone 4-cyclohexyl, 4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased LMP and cell death. This mechanism has critical implications for drug-targeting in MDR tumors where a stressful micro-environment exists.
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Interleukin 1{beta} regulation of the system xc- substrate-specific subunit, xCT, in primary mouse astrocytes involves the RNA-binding protein HuR [Cell Biology]

November 24th, 2015 by Shi, J., He, Y., Hewett, S. J., Hewett, J. A.

System xc- is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the central nervous system (CNS), where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc- in CNS astrocytes secondary to an upregulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'UTR identified numerous copies of adenine- and uridine-rich elements (AREs), raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β- mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels as well as a functional increase in system xc- activity. All together, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.
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RNA Editing Modulates Human Hepatic Aryl Hydrocarbon Receptor Expression by Creating MicroRNA Recognition Sequence [RNA]

November 24th, 2015 by

Adenosine to inosine (A-to-I) RNA editing is the most frequent type of post-transcriptional nucleotide conversion in humans, and it is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. In this study, we investigated the effect of RNA editing on human aryl hydrocarbon receptor (AhR) expression because the AhR transcript potentially forms double-stranded structures, which are targets of ADAR enzymes. In human hepatocellular carcinoma-derived Huh-7 cells, the ADAR1 knockdown reduced the RNA editing levels in the 3′-untranlated region (3′-UTR) of the AhR transcript and increased the AhR protein levels. The ADAR1 knockdown enhanced the ligand-mediated induction of CYP1A1, a gene downstream of AhR. We investigated the possibility that A-to-I RNA editing creates miRNA targeting sites in the AhR mRNA, and found that the miR-378-dependent down-regulation of AhR was abolished by ADAR1 kockdown. These results indicated that the ADAR1-mediated down-regulation of AhR could be attributed to the creation of a miR-378 recognition site in the AhR 3′-UTR. The interindividual differences in the RNA editing levels within the AhR 3′-UTR in a panel of 32 human liver samples were relatively small, whereas the differences in ADAR1 expression were large (220-fold). In the human liver samples, a significant inverse association was observed between the miR-378 and AhR protein levels, suggesting that the RNA editing-dependent down-regulation of AhR by miR-378 contributes to the variability in the constitutive hepatic expression of AhR. In conclusion, this study uncovered, for the first time, that A-to-I RNA editing modulates the potency of xenobiotic metabolism in the human liver.

Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast [Protein Synthesis and Degradation]

November 24th, 2015 by

Despite multiple evidence of the involvement of the proteasome-ubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by Reverses SPT-Phenotype 5 (RSP5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30°C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature, in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway.

Design and Optimization of Anti-Amyloid Domain Antibodies Specific for {beta}-Amyloid and Islet Amyloid Polypeptide [Protein Structure and Folding]

November 24th, 2015 by

Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer's peptide (Aβ). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (VH) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic Aβ42 peptide segment (Aβ residues 17-42) in CDR3 on the solubility and conformational specificity of the corresponding VH domains. We find that VH expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, while other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine and arginine). Moreover, Aβ VH domains with negatively charged CDR3 mutations show significant preference for recognizing Aβ fibrils relative to Aβ monomer, while the same VH domains with other polar CDR3 mutations recognize both Aβ conformers. We observe similar behavior for a VH domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8-37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.
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What can the kinetics of amyloid fibril formation tell about off-pathway aggregation? [Molecular Biophysics]

November 24th, 2015 by

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. While the pathogenic role of these fibrils is not totally established, increasing evidences suggest off-pathway aggregation (OPA) as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step ahead towards the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of OPA relatively to amyloid fibrillization; these include non-linear semi-log plots of amyloid progress curves, highly variable endpoint signals and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of Dynamic Light Scattering (DLS), Circular Dichroism (CD) and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

Nuclear factor of activated T cells-dependent downregulation of the transcription factor GLI1 underlies the growth inhibitory properties of arachidonic acid* [Cell Biology]

November 24th, 2015 by

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis, and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1 and 4-1BB, in AA-treated cells. We demonstrated that downregulation of the transcription factor GLI1 in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1 and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules, and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and downregulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating NFATc1 nuclear translocation, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants reduced the AA-induced apoptosis, downregulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for the future PUFA-based therapeutic approaches.
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