Activation of Toll-like Receptor (TLR) 4 Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress [Metabolism]

September 14th, 2015 by Okla, M., Wang, W., Kang, I., Pashaj, A., Carr, T., Chung, S.

Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study, we hypothesized that TLR4 activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat (HF) diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by HF diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of ER stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced upregulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone, conferred a resistance to the LPS-induced suppression of adaptive thermogenesis. Collectively, our data implicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective BAT activation.

Structure of Human B12 trafficking protein Cb1D reveals molecular mimicry and identifies a new subfamily of Nitro-FMN reductases [Enzymology]

September 14th, 2015 by Yamada, K., Gherasim, C., Banerjee, R., Koutmos, M.

In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. The methylmalonic aciduria and homocystinuria type D protein (MMADHC) commonly referred to as CblD is a key chaperone involved in intracellular cobalamin trafficking and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C terminal domain of human CblD, which is sufficient for its interaction with the methylmalonic aciduria and homocystinuria type C protein (MMADHC) or CblC and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+β fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC and the activation domain of methionine synthase share several distinguishing features and together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function.
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Cystatin D Locates in the Nucleus at Sites of Active Transcription and Modulates Gene and Protein Expression [Glycobiology and Extracellular Matrices]

September 13th, 2015 by

Cystatin D is an inhibitor of lysosomal and secreted cysteine proteases. Strikingly, cystatin D has been found to inhibit proliferation, migration and invasion of colon carcinoma cells indicating tumor suppressor activity that is unrelated to protease inhibition. Here, we demonstrate that a proportion of cystatin D locates within the cell nucleus at specific transcriptionally active chromatin sites. Consistently, transcriptomic analysis show that cystatin D alters gene expression, including that of genes encoding transcription factors such as RUNX1, RUNX2, and MEF2C in HCT116 cells. In concordance with transcriptomic data, quantitative proteomic analysis identified 292 proteins differentially expressed in cystatin D-expressing cells involved in cell adhesion, cytoskeleton, and RNA synthesis and processing. Furthermore, using cytokine arrays we found that cystatin D reduces the secretion of several protumor cytokines such as fibroblast growth factor (FGF)-4, CX3CL1/fractalkine, neurotrophin (NTF)4/NT-4, oncostatin (OSM)-M, pulmonary and activation-regulated chemokine (PARC)/CCL18 and transforming growth factor (TGF)B3/TGF-b3. These results support an unanticipated role of cystatin D in the cell nucleus, controlling the transcription of specific genes involved in crucial cellular functions, which may mediate its protective action in colon cancer.
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Deletion of Muscle-enriched A-type Lamin-Interacting Protein (MLIP) leads to Cardiac Hyperactivation of Akt/mTOR and Impaired Cardiac Adaptation [Signal Transduction]

September 10th, 2015 by

Aging and diseases generally result from tissues inability to maintain homeostasis through adaptation. The adult heart is particularly vulnerable to disequilibrium in homeostasis as its regenerative abilities are limited. Here, we report that Muscle enriched A-type lamin interacting protein (MLIP), a unique protein of unknown function, is required for proper cardiac adaptation. Mlip-/- mice exhibited normal cardiac function despite myocardial metabolic abnormalities and cardiac-specific overactivation of Akt/mTOR pathways. Cardiac-specific MLIP overexpression led to an inhibition of Akt/mTOR, providing evidence of a direct impact of MLIP on these key signaling pathways. Mlip-/- hearts showed an impaired capacity to adapt to stress (isoproterenol-induced hypertrophy), likely due to deregulated Akt/mTOR activity. Genome Wide Association Studies showed a genetic association between Mlip and early response to cardiac stress, supporting MLIP's role in cardiac adaptation. Together, these results revealed that MLIP is required for normal myocardial adaptation to stress through integrated regulation of the Akt/mTOR pathways.
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Glyceraldehyde-3-Phosphate Dehydrogenase Aggregates Accelerate Amyloid-{beta} Amyloidogenesis in Alzheimer Disease [Molecular Bases of Disease]

September 10th, 2015 by

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress, and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome-c from mitochondria. Finally, in the 3xTg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD.
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Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2) [Signal Transduction]

September 10th, 2015 by

JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domains; a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2 demonstrating its central regulatory function. JH2s were considered catalytically inactive but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP-binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with ATP-γS and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, while the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications.

Shedding of endogenous Interleukin-6 receptor (IL-6R) is governed by ADAM proteases while a full-length IL-6R isoform localizes to circulating microvesicles [Immunology]

September 10th, 2015 by

Generation of the soluble Interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine Interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings - or even in vivo - remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.
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Structural insights into cargo recognition by the yeast PTS1-receptor [Protein Structure and Folding]

September 10th, 2015 by

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 μM. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high-resolution mass spectrometry. Most cross-links were found with TPR6, TPR7 as well as the 7C-loop of Pex5p. Surface plasmon resonance (SPR) analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD of the interaction of truncated Pcs60p and Pex5p was in a range of 7.7 μM. ITC and SPR measurements revealed monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept: firstly, cargo recognition and initial tethering by the PTS1-receptor Pex5p and secondly lock-in of receptor and cargo.

Eisosomes regulate PI(4,5)P2 cortical clusters and MAP kinase signaling upon osmotic stress [Signal Transduction]

September 10th, 2015 by Kabeche, R., Madrid, M., Cansado, J., Moseley, J. B.

Eisosomes are multi-protein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P2 regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast S. pombe. Activation of the MAPK Pmk1 was impaired by mutations in the PI(5)-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P2 clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI(5)-kinase Its3, and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P2 clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.

The Q-Soluble-N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-Associated Membrane Proteins (VAMPs) [Cell Biology]

September 10th, 2015 by

SNAREs constitute the core machinery of intracellular membrane fusion but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with TGN VAMP4 and post-Golgi VAMPs 7 and 8. SNAP-47 also interacted with ER and Golgi Syntaxin 5 and with Syntaxin 1 in the absence of Munc18a, when Syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway.
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