The regulation by light of the composition of the photosynthetic apparatus was investigated in photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Leaf chlorophyll, photosynthesis, photosystem II function, and ribulose-1, 5-bisphosphate carboxylase-oxygenase and photosystem II contents were determined for plants grown under high- or low-irradiance growth regimes. Although certain mutant lines had altered chloroplast composition compared to the wild type, all photoreceptor mutants tested were capable of light-dependent changes in chloroplast composition and photosynthetic function, indicating that photoreceptors do not play a central role in the regulation of acclimation at the level of the chloroplast. However, the clear acclimation defect in a det1 signal transduction mutant indicates that photoreceptor-controlled responses either share regulatory components with acclimation, or are important in the expression of components which in turn regulate acclimation. We suggest that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response.

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans.

Loss of muscle mass via protein degradation is an important clinical problem but we know little of how muscle protein degradation is regulated genetically. To gain insight our labs developed C. elegans into a model for understanding the regulation of muscle protein degradation. Past studies uncovered novel functional roles for genes affecting muscle and/or involved in signalling in other cells or tissues. Here we examine most of the genes previously identified as the sites of mutations affecting muscle for novel roles in regulating degradation. We evaluate genomic (RNAi knockdown) approaches and combine them with our established genetic (mutant) and pharmacologic (drugs) approaches to examine these 159 genes. We find that RNAi usually recapitulates both organismal and sub-cellular mutant phenotypes but RNAi, unlike mutants, can frequently be used acutely to study gene function solely in differentiated muscle. In the majority of cases where RNAi does not produce organismal level phenotypes, sub-cellular defects can be detected; disrupted proteostasis is most commonly observed. We identify 48 genes in which mutation or RNAi knockdown causes excessive protein degradation; myofibrillar and/or mitochondrial morphologies are also disrupted in 19 of these 48 cases. These 48 genes appear to act via at least three sub-networks to control bulk degradation of protein in muscle cytosol. Attachment to the extracellular matrix regulates degradation via unidentified proteases and affects myofibrillar and mitochondrial morphology. Growth factor imbalance and calcium overload promote lysosome based degradation whereas calcium deficit promotes proteasome based degradation, in both cases myofibrillar and mitochondrial morphologies are largely unaffected. Our results provide a framework for effectively using RNAi to identify and functionally cluster novel regulators of degradation. This clustering allows prioritization of candidate genes/pathways for future mechanistic studies.

Host defense against infection is induced by Toll-like and interleukin (IL)-1 receptors, and controlled by the transcription factor NF-κB. Our earlier studies have shown that IL-1 activation impacts cytoskeletal structure and that IL-1 receptor (IL-1RI) function is substrate-dependent. Here we identify a novel regulatory component, TILRR, which amplifies activation of IL-1RI and coordinates IL-1-induced control with mechanotransduction. We show that TILRR is a highly conserved and widely expressed enhancer of IL-1-regulated inflammatory responses and, further, that it is a membrane-bound glycosylated protein with sequence homology to members of the FRAS-1 family. We demonstrate that TILRR is recruited to the IL-1 receptor complex and magnifies signal amplification by increasing receptor expression and ligand binding. In addition, we show that the consequent potentiation of NF-κB is controlled through IL-1RI-associated signaling components in coordination with activation of the Ras GTPase. Using mutagenesis, we demonstrate that TILRR function is dependent on association with its signaling partner and, further, that formation of the TILRR-containing IL-1RI complex imparts enhanced association of the MyD88 adapter during ligand-induced activation of NF-κB. We conclude that TILRR is an IL-1RI co-receptor, which associates with the signaling receptor complex to enhance recruitment of MyD88 and control Ras-dependent amplification of NF-κB and inflammatory responses.

In common with many other higher plant species, Arabidopsis undergoes photosynthetic acclimation, altering the composition of the photosynthetic apparatus in response to fluctuations in its growth environment. The changes in photosynthetic function that result from acclimation can be detected in a noninvasive manner by monitoring chlorophyll (Chl) fluorescence. This technique has been used to develop a screen that enables the rapid identification of plants defective atACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT(APE) loci. The application of this screen to a population of T-DNA-transformed Arabidopsis has successfully led to the identification of a number of mutant lines with altered Chl fluorescence characteristics. Analysis of photosynthesis and pigment composition in leaves from three such mutants showed that they had altered acclimation responses to the growth light environment, each having a distinct acclimation-defective phenotype, demonstrating that screening for mutants using Chl fluorescence is a viable strategy for the investigation of acclimation. Sequencing of the genomic DNA flanking the T-DNA elements showed that in the ape1mutant, a gene was disrupted that encodes a protein of unknown function but that appears to be specific to photosynthetic organisms, whereas the ape2 mutant carries an insertion in the region of the TPT gene encoding the chloroplast inner envelope triose phosphate/phosphate translocator.

Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity.

Aims: Cells have developed quality control systems for protection against proteotoxicity. Misfolded and aggregation-prone proteins, which are behind the initiation and progression of many neurodegenerative diseases (ND), are known to challenge the proteostasis network of the cells. We aimed to explore the role of DNJ-27/ ERdj5, an endoplasmic reticulum (ER)-resident thioredoxin protein required as a disulfide reductase for the degradation of misfolded proteins, in well-established Caenorhabditis elegans models of Alzheimer, Parkinson and Huntington diseases. Results: We demonstrate that DNJ-27 is an ER luminal protein and that its expression is induced upon ER stress via IRE-1/XBP-1. When dnj-27 expression is downregulated by RNA interference we find an increase in the aggregation and associated pathological phenotypes (paralysis and motility impairment) caused by human b-amyloid peptide (Ab), a-synuclein (a-syn) and polyglutamine (polyQ) proteins. In turn, DNJ-27 overexpression ameliorates these deleterious phenotypes. Surprisingly, despite being an ER-resident protein, we show that dnj-27 downregulation alters cytoplasmic protein homeostasis and causes mitochondrial fragmentation. We further demonstrate that DNJ-27 overexpression substantially protects against the mitochondrial fragmentation caused by human Ab and a-syn peptides in these worm models. Innovation: We identify C. elegans dnj-27 as a novel protective gene for the toxicity associated with the expression of human Ab, a-syn and polyQ proteins, implying a protective role of ERdj5 in Alzheimer, Parkinson and Huntington diseases. Conclusion: Our data support a scenario where the levels of DNJ-27/ERdj5 in the ER impact cytoplasmic protein homeostasis and the integrity of the mitochondrial network which might underlie its protective effects in models of proteotoxicity associated to human ND. Antioxid. Redox Signal. 20,[217][218][219][220][221][222][223][224][225][226][227][228][229][230][231][232][233][234][235]

Haemoglobins are iron-containing proteins that transport oxygen in the blood of most vertebrates. The mitochondrion is the cellular organelle which consumes oxygen in order to synthesise ATP. Mitochondrial dysfunction is implicated in neurodegeneration and ageing. We find that α and β haemoglobin (Hba and Hbb) proteins are altered in their distribution in mitochondrial fractions from degenerating brain. We demonstrate that both Hba and Hbb are co-localised with the mitochondrion in mammalian brain. The precise localisation of the Hbs is within the inner membrane space and associated with inner mitochondrial membrane. Relative mitochondrial to cytoplasmic ratios of Hba and Hbb show changing distributions of these proteins during the process of neurodegeneration in the pcd5j mouse brain. A significant difference in mitochondrial Hba and Hbb content in the mitochondrial fraction is seen at 31 days after birth, this corresponds to a stage when dynamic neuronal loss is measured to be greatest in the Purkinje Cell Degeneration mouse. We also report changes in mitochondrial Hba and Hbb levels in ageing brain and muscle. Significant differences in mitochondrial Hba and Hbb can be seen when comparing aged brain to muscle, suggesting tissue specific functions of these proteins in the mitochondrion. In muscle there are significant differences between Hba levels in old and young mitochondria. To understand whether the changes detected in mitochondrial Hbs are of clinical significance, we examined Parkinson's disease brain, immunohistochemistry studies suggest that cell bodies in the substantia nigra accumulate mitochondrial Hb. However, western blotting of mitochondrial fractions from PD and control brains indicates significantly less Hb in PD brain mitochondria. One explanation could be a specific loss of cells containing mitochondria loaded with Hb proteins. Our study opens the door to an examination of the role of Hb function, within the context of the mitochondrion—in health and disease.