Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental and theoretical IR spectroscopy [Protein Structure and Folding]

August 13th, 2015 by

Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR-difference spectroscopy and QM/MM simulations we elucidate that the Mg2+ coordination by the phosphate groups, which varies largely among the X-ray structures, is the same for Ran and Ras. A new X-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg2+ in GTPases. The Mg2+ coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr39 side chain of Ran between the γ-phosphate and Gln69 prevents the optimal positioning of the attacking water molecule by the Gln69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr25 to the α-phosphate in Ran. By integration of X-ray structure analysis, experimental and theoretical IR spectroscopy the catalytic center of the X-ray structural models are further refined to sub-Ångstrom resolution, allowing an improved understanding of catalysis.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental and theoretical IR spectroscopy [Protein Structure and Folding]

A novel basal body protein and Polo-like kinase substrate is required for basal body segregation and flagellum adhesion in Trypanosoma brucei [Microbiology]

August 13th, 2015 by Hu, H., Zhou, Q., Li, Z.

The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confines TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of the bilobe resident TbCentrin2. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on A novel basal body protein and Polo-like kinase substrate is required for basal body segregation and flagellum adhesion in Trypanosoma brucei [Microbiology]

Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content [Membrane Biology]

August 13th, 2015 by

Cancer cells emit extracellular vesicles (EVs) containing unique molecular signatures. Here we report that oncogenic EGF receptor (EGFR) and its inhibitors reprogram phosphoproteomes and cargo of tumor cell derived EVs. Thus, phosphorylated EGFR (P-EGFR) and several other receptor tyrosine kinases (RTKs) can be detected in EVs purified from plasma of tumor bearing mice and from conditioned media of cultured cancer cells. Treatment of EGFR-driven tumor cells with second generation EGFR kinase inhibitors (EKIs), including CI-1033 and PF-00299804, but not with anti-EGFR antibody (Cetuximab), or etoposide triggers a burst in emission of exosome-like EVs containing EGFR, P-EGFR and genomic DNA (exo-gDNA). The EV release can be attenuated by treatment with inhibitors of exosome biogenesis (GW4869) and caspase pathways (ZVAD). The content of P-EGFR isoforms (Y845, Y1068 and Y1173), ERK and AKT varies between cells and their corresponding EVs, and as a function of EKI treatment. Immunocapture experiments reveal the presence of EGFR and exo-gDNA within the same EV population following EKI treatment. These findings suggest that targeted agents may induce cancer cells to change the EV emission profiles reflective of drug-related therapeutic stress. We suggest that EV-based assays may serve as companion diagnostics for targeted anticancer agents.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content [Membrane Biology]

Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4{alpha} [Metabolism]

August 13th, 2015 by

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein (apo)B-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short-term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long-term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long-term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Microprocessor Complex Subunit DiGeorge Syndrome Critical Region Gene 8 (Dgcr8) is Required for Schwann Cell Myelination and Myelin Maintenance [Gene Regulation]

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.

ERK2-PYRUVATE KINASE AXIS PERMITS PMA-INDUCED MEGAKARYOCYTE DIFFERENTIATION IN K562 CELLS [Gene Regulation]

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.