Self-assembly is prerequisite for catalysis of Fe(II) oxidation by catalytically active subunits of ferritin [Protein Structure and Folding]

September 14th, 2015 by Honarmand Ebrahimi, K., Hagedoorn, P.-L., Hagen, W. R.

Fe(III)-storage by Ferritin is an essential process of the iron homeostasis machinery. It begins by translocation of Fe(II) from outside the hollow spherical-shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a diiron binding site, the ferroxidase center, buried in the middle of each active subunit. The pathway of Fe(II) to the ferroxidase center has remained elusive, and the importance of self-assembly for the functioning of the ferroxidase center has not been investigated. Here we report spectroscopic and metal-ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution. We show that in this mutant metal ion binding to the ferroxidase center and Fe(II) oxidation at this site were obliterated. However, metal-ion binding to a conserved third site (site C), which is located in the inner surface of each subunit in the vicinity of the ferroxidase center and is believed to be the path for Fe(II) to the ferroxidase center, was not disrupted. These results are the basis of a new model for Fe(II) translocation to the ferroxidase center: self-assembly creates channels that guide the Fe(II) ions towards the ferroxidase center directly through the protein shell, and not via the internal cavity and site C. The results may be of significance for understanding the molecular basis of ferritin-related disorders such as neuroferritinopathy in which the 24-meric structure with 432 symmetry is distorted.

Resistance of Dynamin-related protein 1 Oligomers to Disassembly Impairs Mitophagy Resulting in Myocardial Inflammation and Heart failure [Metabolism]

September 14th, 2015 by

We previously reported that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy (DCM). The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python DCM model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast (MEF) model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F MEFs were depolarized and had reduced calcium uptake, with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age, and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis and activation of sterile myocardial inflammation resulting in heart failure.
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A Kissing-Loop is Important for BtuB Riboswitch Ligand Sensing and Regulatory Control [Gene Regulation]

September 14th, 2015 by Lussier, A., Bastet, L., Chauvier, A., Lafontaine, D. A.

RNA-based genetic regulation is exemplified by metabolite-binding riboswitches that modulate gene expression through conformational changes. Crystal structures show that the Escherichia coli btuB riboswitch contains a kissing-loop interaction that is in close proximity to the bound ligand. To analyze the role of the kissing-loop interaction in the riboswitch regulatory mechanism, we used RNase H cleavage assays to probe the structure of nascent riboswitch transcripts produced by the E. coli RNA polymerase. By monitoring the folding of the aptamer, kissing-loop and riboswitch expression platform, we established the conformation of each structural component in the absence or presence of bound adenosylcobalamin. We found that the kissing-loop interaction is not essential for ligand binding. However, we showed that kissing-loop formation improves ligand binding efficiency and is required to couple ligand binding to the riboswitch conformational changes involved in regulating gene expression. These results support a mechanism by which the btuB riboswitch modulates the formation of a tertiary structure to perform metabolite sensing and regulate gene expression.

AMP-activated Protein Kinase Stimulates Warburg-Like Glycolysis and Activation of Satellite Cells during Muscle Regeneration [Metabolism]

September 14th, 2015 by Fu, X., Zhu, M.-J., Dodson, M. V., Du, M.

Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent, but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a non-canonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating non-canonical Shh promote proliferation of satellite cells, which is abolished due to satellite cell-specific AMPKα1 knockout. Taken together, AMPKα1 is a critical mediator linking non-canonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.

Robust glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI family member protein is critical for oxidative stress resistance in Saccharomyces cerevisiae [Protein Synthesis and Degradation]

September 14th, 2015 by

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. Accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, and leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, but their functions are largely dependent on glutathione cofactor whose availability is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein; a member of ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae plays an indispensable role in regulating redox homeostasis. Our results highlight that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared to another paralog, Hsp34. On the other hand, glyoxalase defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. In addition to that, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress. Besides, Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1 which is implicated in the familial form of Parkinson disease complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying importance of these proteins in the maintenance of ROS homeostasis across phylogeny.
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Effect of Cholesterol Reduction on Receptor Signaling in Neurons [Neurobiology]

September 14th, 2015 by Fukui, K., Ferris, H. A., Kahn, C. R.

Diabetes mellitus is associated with a variety of complications, including alterations in the central nervous system (CNS). We have recently shown that diabetes results in a reduction of cholesterol synthesis in the brain due to decreased insulin stimulation of SREBP2 mediated cholesterol synthesis in neuronal and glial cells. In the present study, we have explored the effects of the decrease in cholesterol on neuronal cell function using GT1-7 hypothalamic cells subjected to cholesterol depletion in vitro using three independent methods: 1) exposure to methyl-beta-cyclodextrin (MBCD), 2) treatment with the HMG-CoA reductase inhibitor simvastatin and 3) shRNA-mediated knockdown of SREBP2. All three methods produced 20-31% reductions in cellular cholesterol content, similar to the decrease in cholesterol synthesis observed in diabetes. All cholesterol-depleted neuron-derived cells, independent of the method of reduction, exhibited decreased phosphorylation/ activation of IRS-1 and Akt following stimulation by insulin, IGF-1 or the neurotrophins (NGF and BDNF). ERK phosphorylation/activation was also decreased after MBCD and statin treatment, but increased in cells following SREBP2 knockdown. In addition, apoptosis in the presence of amyloid-β; was increased. Reduction in cellular cholesterol also resulted in increased basal autophagy and impairment of induction of autophagy by glucose deprivation. Together, these data indicate that a reduction in neuron-derived cholesterol content, similar to that observed in diabetic brain, creates a state of insulin and growth factor resistance that could contribute to CNS-related complications of diabetes, including increased risk of neurodegenerative diseases such as Alzheimer′s disease.

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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