Microprocessor Complex Subunit DiGeorge Syndrome Critical Region Gene 8 (Dgcr8) is Required for Schwann Cell Myelination and Myelin Maintenance [Gene Regulation]

August 13th, 2015 by Lin, H.-P., Oksuz, I., Hurley, E., Wrabetz, L., Awatramani, R.

We investigated the role of a key component of the Microprocessor complex, DGCR8, in the regulation of myelin formation and maintenance. We found that conditionally ablating Dgcr8 in Schwann cells (SCs) during development results in an arrest of SC differentiation. Dgcr8 conditional knockout (cKO) SCs fail to form 1:1 relationships with axons or, having achieved this, fail to form myelin sheaths. The expression of genes normally found in immature SCs, such as sex determining region Y-box 2 (Sox2), is increased in Dgcr8 cKO SCs, while the expression of myelin-related genes, including the master regulatory transcription factor early growth response 2 (Egr2), is decreased. Additionally, expression of a novel gene expression program involving sonic hedgehog (Shh), activated de novo in injured nerves, is elevated in Dgcr8 cKOs, but not in Egr2 null mice, a model of SC differentiation arrest, suggesting that the injury-related gene expression program in Dgcr8 cKOs cannot be attributed to differentiation arrest. Inducible ablation of Dgcr8 in adult SCs results in gene expression changes similar to those found in cKOs, including an increase in the expression of Sox2 and Shh. Analyses of these nerves mainly reveal normal myelin thickness and axon size distribution, but some dedifferentiated SCs and increased macrophage infiltration. Together, our data suggests that Dgcr8 is responsible for modulation of gene expression programs underlying myelin formation and maintenance, as well as suppression of an injury-related gene expression program.
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Conserved overlapping gene arrangement, restricted expression and biochemical activities of DNA polymerase {nu}; (POLN) [Enzymology]

August 12th, 2015 by

DNA polymerase ν (POLN) is one of 16 DNA polymerases encoded in vertebrate genomes. It is important to determine its gene expression patterns, biological roles, and biochemical activities. By quantitative analysis of mRNA expression we found that POLN from the zebrafish Danio rerio is expressed predominantly in testis. POLN is not detectably expressed in zebrafish embryos or in mouse embryonic stem cells. Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development. Analysis of transcripts revealed that vertebrate POLN has an unusual gene expression arrangement, sharing a first exon with HAUS3, the gene encoding augmin-like complex subunit 3. HAUS3 is broadly expressed in embryonic and adult tissues, in contrast to POLN. Differential expression of POLN and HAUS3 appears to arise by alternate splicing of transcripts in mammalian cells and zebrafish. When POLN was ectopically overexpressed in human cells, it specifically coimmunoprecipitated with the homologous recombination factors BRCA1 and FANCJ, but not with previously suggested interaction partners (HELQ and members of the Fanconi anemia core complex). Purified zebrafish POLN protein is capable of thymine glycol (Tg) bypass and strand displacement, with activity dependent on a basic amino acid residue known to stabilize the primer-template. These properties are conserved with the human enzyme. Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase.


August 12th, 2015 by

Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA) induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with upregulated expression of two PK isoforms (PKM2 and PKR) in an ERK2 dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behaviour. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. Besides metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the upregulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

Signal transduction and intracellular trafficking by the interleukin 36 receptor [Signal Transduction]

August 12th, 2015 by

Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor (IL-1R) family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.

Neutrophil Elastase promotes Interleukin-1{beta} secretion from Human Coronary Endothelium [Immunology]

August 12th, 2015 by

The endothelium is critically involved in the pathogenesis of atherosclerosis by producing proinflammatory mediators, including interleukin-1 beta (IL-1β). Coronary arteries from patients with ischaemic heart disease express large amounts of IL-1β in endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1β remains to be elucidated. We investigated neutrophil elastase (NE), a potent serine protease detected in vulnerable areas of human carotid plaques, as a potential ′trigger′ for IL-1β processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1β from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1β in ECs and causes significant secretion of bioactive IL-1β via extracellular vesicles. This release was significantly attenuated by inhibition of neutrophil elastase, but not caspase-1. Transient increases in intracellular Ca2+ levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1β was detected within LAMP-1 positive multivesicular bodies (MVBs). The released vesicles contained bioactive IL-1β. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1β. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1β secretion from ECs; this could reveal additional potential anti-IL-1β therapeutic targets and provide further insight into the inflammatory process by which vascular disease develops.

Reaction of hydrogen sulfide with disulfide and sulfenic acid to form the strongly nucleophilic persulfide [Metabolism]

August 12th, 2015 by

Hydrogen sulfide (H2S) is increasingly recognized to modulate physiological processes in mammals through mechanisms that are currently under scrutiny. H2S is not able to react with reduced thiols (RSH). However, H2S -precisely, HS-- is able to react with oxidized thiol derivatives. We performed a systematic study of the reactivity of HS- toward symmetric low molecular weight disulfides (RSSR) and mixed albumin (HSA) disulfides. Correlations with thiol acidity and computational modeling showed that the reaction occurs through a concerted mechanism. Comparison to analogous reactions of thiolates indicated that the intrinsic reactivity of HS- is one order of magnitude lower than that of thiolates. In addition, H2S is able to react with sulfenic acids (RSOH). The rate constant of the reaction of H2S with the sulfenic acid formed in HSA was determined. Both reactions of H2S with disulfides and sulfenic acids yield persulfides (RSSH), recently identified post-translational modifications. The formation of this derivative in HSA was determined and the rate constants of its reactions with a reporter disulfide and with peroxynitrite revealed that persulfides are better nucleophiles than thiols, consistent with the alpha effect. Experiments with cells in culture showed that treatment with hydrogen peroxide enhanced the formation of persulfides. Biological implications are discussed. Our results give light on the mechanisms of persulfide formation and provide quantitative evidence for the high nucleophilicity of these novel derivatives, setting the stage for understanding the contribution of the reactions of H2S with oxidized thiol derivatives to H2S effector processes.

Modulator of Apoptosis 1 (MOAP-1) is a tumor suppressor protein linked to RASSF1A [Cell Biology]

August 12th, 2015 by

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein, Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein is yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA downregulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor, MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and upregulation of genes involved in cancer regulatory pathways that includes apoptosis (p53, Fas and MST1), DNA damage control (PARP and ATM), those within the cell metabolism (IR-alpha, IR-beta and AMPK) and a stablizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor dependent pathway and drive tumorigenesis.

Polymorphic variants of human rhodanese exhibit differences in thermal stability and sulfurtransfer kinetics [Enzymology]

August 12th, 2015 by Libiad, M., Sriraman, A., Banerjee, R.

Rhodanese is a component of the mitochondrial hydrogen sulfide (H2S) oxidation pathway. Rhodanese catalyzes the transfer of sulfane sulfur from glutathione persulfide (GSSH) to sulfite generating thiosulfate and from thiosulfate to cyanide generating thiocyanate. Two polymorphic variations have been identified in the rhodanese coding sequence in the French Caucasian population. The first, 306A→C, has an allelic frequency of 1 percent and results in an E102D substitution in the encoded protein. The second polymorphism 853C→G, has an allelic frequency of 5 percent and leads to a P285A substitution. In this study, we have examined differences in the stability between wild-type rhodanese and the E102D and P285A variants and in the kinetics of the sulfurtransfer reactions. The Asp102 and Ala285 variants are more stable than wild-type rhodanese and exhibit kcat/KM,CN values that are 17 and 1.6 fold higher, respectively. All three rhodanese forms preferentially catalyze sulfurtransfer from GSSH to sulfite, generating thiosulfate and glutathione. The kcat/KM,sulfite values for the variants in the sulfurtransfer reaction from GSSH to sulfite were 1.6 (Asp102) and 4 fold (Ala285) lower than for wild-type rhodanese while the kcat/KM,GSSH values were similar for all three enzymes. Thiosulfate-dependent H2S production in murine liver lysate is low, consistent with a role for rhodanese in sulfide oxidation. Our studies show that polymorphic variations that are distant from the active site differentially modulate the sulfurtransferase activity of human rhodanese to cyanide versus sulfite and might be important in differences in susceptibility to diseases where rhodanese dysfunction has been implicated, e.g. inflammatory bowel diseases.

Fibroblast-Derived Neuregulin-1 Promotes Compensatory ErbB3 Signaling in Mutant BRAF Melanoma [Cell Biology]

August 12th, 2015 by Capparelli, C., Rosenbaum, S., Berger, A. C., Aplin, A. E.

RAF inhibitors are first-line treatments for patients harboring V600E/K mutant BRAF melanoma. While RAF inhibitors produce high response rates, the degree of tumor regression is heterogeneous. Compensatory/adaptive responses to targeted inhibitors are frequently initiated by the activation of growth factor receptor tyrosine kinases (RTKs) including ErbB3, and factors from the tumor microenvironment may play an important role. We have previously shown that mutant BRAF melanoma cells have enhanced activation of ErbB3 following RAF inhibition; however, the source of neuregulin 1 (NRG1), the ligand for ErbB3, is unknown. In this study, we demonstrate that NRG1 is highly expressed by dermal fibroblasts and cancer associated fibroblasts (CAFs) isolated from mutant BRAF melanomas. Conditioned medium from fibroblasts and CAFs enhanced ErbB3 pathway activation and limited RAF inhibitor cytotoxicity in V600 mutant BRAF-harboring melanomas. Targeting the ErbB3/ErbB2 pathway partially reversed the protective effects of fibroblast/CAF-derived NRG1 on cell growth properties of RAF inhibitor-treated melanoma cells. These findings support the idea that NRG1, acting in a paracrine manner, promotes resistance to RAF inhibitors and emphasize that targeting the ErbB3/ErbB2 pathway will likely improve the efficacy of RAF inhibitors for mutant BRAF melanoma patients.

Structure-functional Characterization of Cytochrome P450 Sterol 14{alpha}-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs [Protein Structure and Folding]

August 12th, 2015 by

Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths per year. The cytochrome P450 (CYP) 51 inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, while gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and X-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min-1, respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The X-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, while the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.
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