Glucosamine modulates T cell differentiation through downregulating N-linked glycosylation of CD25 [Glycobiology and Extracellular Matrices]

October 14th, 2015 by

Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through downregulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both downregulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis.

Complement factor H binds to human serum apolipoprotein E and mediates complement regulation on high-density lipoprotein particles [Immunology]

October 14th, 2015 by

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19-20 (FH19-20) and 5-7 (FH5-7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We have previously shown that inhibition of FH binding by a recombinant FH5-7 construct impairs survival of FH-binding pathogens in human blood. In this study we found that, upon exposure to full blood, addition of FH5-7 reduces survival of surprisingly also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5-7. We found that although FH5-7 does not reduce complement regulation in the actual fluid phase of plasma it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5-7 domains. Furthermore, binding of FH5-7 to HDL was dependent on the concentration of apoE on the HDL-particles. These findings explain why addition of FH5-7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5-7, and regulates alternative pathway activation on plasma HDL particles.
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The inflammasome adaptor ASC induces procaspase-8 death effector domain filaments [Signal Transduction]

October 14th, 2015 by

Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerisation of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs, and represents an unusual heterotypic interaction between domains of the death-fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death-fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors.

The Polycomb Repressive Complex 1 Protein BMI1 is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells [Genomics and Proteomics]

October 14th, 2015 by Abdouh, M., Hanna, R., El Hajjar, J., Flamier, A., Bernier, G.

The Polycomb Repressive Complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitylates histone H2A (H2Aub) and is associated with silenced developmental genes at facultative heterochromatin. It is however assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1 and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2Aub deposition, constitutive heterochromatin formation and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome
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Subunits of ADA-Two-A-Containing (ATAC) or Spt-Ada-Gcn5-Acetyltrasferase (SAGA) Coactivator Complexes Enhance the Acetyltransferase Activity of GCN5 [Enzymology]

October 14th, 2015 by

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT family of HATs. In metazoans, this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 Acetyltransferase) and ATAC (Ada Two A Containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, while in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) GCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it is part of purified recombinant hATAC or hSAGA HAT modules, or endogenous hATAC or hSAGA complexes, using histone tail peptides and full-length histones as substrates. We demonstrate that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest stronger influence of ADA2b as compared to ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones is not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules, and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes.
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The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains is Governed by their NH2-terminal and Internal Protein Regions [Metabolism]

October 14th, 2015 by Park, J. W., Reed, J. R., Backes, W. L.

In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our lab has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N-terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302 peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations but was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo.
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JNK associated leucine zipper protein functions as a docking platform for Polo like kinase 1 and regulation of the associating transcription factor Forkhead box protein K1 [Signal Transduction]

October 14th, 2015 by

JLP (JNK associated Leucine zipper protein) is a scaffolding protein that interacts with various signaling proteins associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation and apoptosis. Here we identified Polo like kinase 1 (PLK1) as a novel interaction partner of JLP through mass spectrometric approaches. Our results indicate that JLP is phospho-primed by PLK1 on Thr 351, which is recognized by the PBD of PLK1 leading to phosphorylation of JLP at additional sites. SILAC and quantitative LC-MS/MS analysis was performed to identify PLK1 dependent JLP interacting proteins. Treatment of cells with the PLK1 kinase inhibitor BI2536 suppressed binding of the Forkhead box protein K1 (FOXK1) transcriptional repressor to JLP. JLP was found to interact with PLK1 and FOXK1 during mitosis. Moreover, knockdown of PLK1 affected the interaction between JLP and FOXK1. FOXK1 is a known transcriptional repressor of the CDK inhibitor p21/WAF1 and knockdown of JLP resulted in increased FOXK1 protein levels and a reduction of p21 transcript levels. Our results suggest a novel mechanism by which FOXK1 protein levels and activity are regulated by associating with JLP and PLK1.
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cAMP-Dependent Protein Kinase (PKA) Signaling is Impaired in the Diabetic Heart [Signal Transduction]

October 14th, 2015 by Bockus, L. B., Humphries, K. M.

Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed β-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Controls and streptozotocin-induced diabetic mice were therefore administered 8Br-cAMP acutely to activate PKA in a receptor-independent manner and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator, PFK-2, was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed remarkably similar changes in PKA signaling, suggesting lipotoxicity is a major contributor to diabetes induced β-adrenergic signaling dysfunction. This work demonstrates PKA signaling responds to metabolic conditions and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function.

Hypoxia inhibits myogenic differentiation through p53-dependent induction of Bhlhe40 [Cell Biology]

October 14th, 2015 by Wang, C., Liu, W., Liu, Z., Chen, L., Liu, X., Kuang, S.

Satellite cells are muscle resident stem cells capable of self-renewal and differentiation to repair injured muscles. However, muscle injury often leads to an ischemic hypoxia environment that impedes satellite cell differentiation and reduces the efficiency of muscle regeneration. Here, we performed microarray analysis and identified the basic Helix-Loop-Helix family transcription factor Bhlhe40 as a candidate mediator of the myogenic inhibitory effect of hypoxia. Bhlhe40 is strongly induced by hypoxia in satellite cell-derived primary myoblasts. Overexpression of Bhlhe40 inhibits Myogenin expression and mimics the effect of hypoxia on myogenesis. Inhibition of Bhlhe40 conversely upregulates Myogenin expression and promotes myogenic differentiation. Importantly, Bhlhe40 knockdown rescues myogenic differentiation under hypoxia. Mechanistically, Bhlhe40 binds to the proximal E-boxes of Myogenin promoter and reduces the binding affinity and transcriptional activity of MyoD on Myogenin. Interestingly, hypoxia induces Bhlhe40 expression independent of HIF1α, but through a novel p53-dependent signaling pathway. Together, our study establishes a crucial role of Bhlhe40 in mediating the repressive effect of hypoxia on myogenic differentiation and suggests that inhibition of Bhlhe40 or p53 may facilitate muscle regeneration after ischemic injuries.

Transthyretin Binding Heterogeneity and Anti-amyloidogenic Activity of Natural Polyphenols and their Metabolites [Molecular Bases of Disease]

October 14th, 2015 by

Transthyretin (TTR) is an amyloidogenic protein, whose amyloidogenic potential is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of X-ray analysis of TTR in complex with both ligands. While revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, while genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites, and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulphate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by X-ray analysis of their complexes with TTR.
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