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Sample records for protein degradation pathway

  1. Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway

    PubMed Central

    Sheehan, Patricia; Zhu, Mei; Beskow, Anne; Vollmer, Cyndel

    2016-01-01

    Synaptic vesicle (SV) pools must maintain a functional repertoire of proteins to efficiently release neurotransmitter. The accumulation of old or damaged proteins on SV membranes is linked to synaptic dysfunction and neurodegeneration. However, despite the importance of SV protein turnover for neuronal health, the molecular mechanisms underlying this process are largely unknown. Here, we have used dissociated rat hippocampal neurons to investigate the pathway for SV protein degradation. We find that neuronal activity drives the degradation of a subset of SV proteins and that the endosomal sorting complex required for transport (ESCRT) machinery and SV-associated GTPase Rab35 are key elements of this use-dependent degradative pathway. Specifically, neuronal activity induces Rab35 activation and binding to the ESCRT-0 protein Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to SV pools, thereby initiating SV protein degradation via the ESCRT pathway. Our findings show that the Rab35/ESCRT pathway facilitates the activity-dependent removal of specific proteins from SV pools, thereby maintaining presynaptic protein homeostasis. SIGNIFICANCE STATEMENT Synaptic transmission is mediated by the release of chemical neurotransmitters from synaptic vesicles (SVs). This tightly regulated process requires a functional pool of SVs, necessitating cellular mechanisms for removing old or damaged proteins that could impair SV cycling. Here, we show that a subset of SV proteins is degraded in an activity-dependent manner and that key steps in this degradative pathway are the activation of the small GTPase Rab35 and the subsequent recruitment of the endosomal sorting complex required for transport (ESCRT) machinery to SV pools. Further, we demonstrate that ESCRT-0 component Hrs is an effector of Rab35, thus providing novel mechanistic insight into the coupling of neuronal activity with SV protein degradation and the

  2. Molecular Pathways: Turning Proteasomal Protein Degradation into a Unique Treatment Approach

    PubMed Central

    Stintzing, Sebastian; Lenz, Heinz-Josef

    2015-01-01

    Cancer treatment regimens have evolved from single cytotoxic substances affecting all proliferative tissues towards antibodies and kinase inhibitors targeting tumor specific pathways. Treatment efficacy and cancer survival has overall improved and side effects have become less frequent. The ubiquitin proteasome system (UPS) mediated proteasomal protein degradation is the most critical pathway to regulate the quantity of signal proteins involved in carcinogenesis and tumor progression. These processes are, as well as protein recycling, highly regulated and offer targets for biomarker and drug development. Unspecific proteasome inhibitors such as bortezomib and carfilzomib have shown clinical efficacy and are approved for clinical use. Inhibitors of more substrate specific enzymes of degradation processes are developed and in early clinical trials. The novel compounds focus on the degradation of key regulatory proteins such as p53, p27Kip1 and β-catenin, and inhibitors specific for growth factor receptor kinases turnover are in pre-clinical testing. PMID:24756373

  3. Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

    ERIC Educational Resources Information Center

    Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

    2006-01-01

    This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

  4. Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

    PubMed Central

    Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

    2012-01-01

    The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

  5. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  6. The Mitochondrial Translocator Protein, TSPO, Inhibits HIV-1 Envelope Glycoprotein Biosynthesis via the Endoplasmic Reticulum-Associated Protein Degradation Pathway

    PubMed Central

    Zhou, Tao; Dang, Ying

    2014-01-01

    ABSTRACT The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4+ T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. IMPORTANCE The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral

  7. Inhibition of protein ubiquitination by paraquat and 1-methyl-4-phenylpyridinium impairs ubiquitin-dependent protein degradation pathways

    PubMed Central

    Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

    2016-01-01

    Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP+, or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ–induced cell death. Inhibition of proteasomal activity by PQ was found to be a late event in cell death progression, and had no effect on either the toxicity of MPP+ or PQ, or the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins) and carbonylated proteins induced by PQ. PQ- and MPP+-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagic. We confirmed that PQ and MPP+ impaired autophagy flux, and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane associated foci in yeast cells. Our results demonstrate that inhibition of protein ubiquitination by PQ and MPP+ is involved in the dysfunction of Ub-dependent protein

  8. Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.

    PubMed

    Rincon-Heredia, Ruth; Flores-Benitez, David; Flores-Maldonado, Catalina; Bonilla-Delgado, José; García-Hernández, Vicky; Verdejo-Torres, Odette; Castillo, Aida M; Larré, Isabel; Poot-Hernández, Augusto C; Franco, Martha; Gariglio, Patricio; Reyes, José L; Contreras, Rubén G

    2014-01-01

    In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

  9. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  10. Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin-proteasome pathway.

    PubMed

    He, Q; Liu, Y

    2005-11-01

    Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) promotes its degradation through the ubiquitin-proteasome pathway. Ubiquitination of FRQ requires FWD-1 (F-box/WD-40 repeat-containing protein-1), which is the substrate-recruiting subunit of an SCF (SKP/Cullin/F-box)-type ubiquitin ligase. In the fwd-1 mutant strains, FRQ degradation is defective, resulting in the accumulation of hyperphosphorylated FRQ and the loss of the circadian rhythmicities. The CSN (COP9 signalosome) promotes the function of SCF complexes in vivo. But in vitro, deneddylation of cullins by CSN inhibits SCF activity. In Neurospora, the disruption of the csn-2 subunit impairs FRQ degradation and compromises the normal circadian functions. These defects are due to the dramatically reduced levels of FWD-1 in the csn-2 mutant, a result of its rapid degradation. Other components of the SCF(FWD-1) complex, SKP-1 and CUL-1 are also unstable in the mutant. These results establish important roles for SCF(FWD-1) and CSN in the circadian clock of Neurospora and suggest that they are conserved components of the eukaryotic circadian clocks. In addition, these findings resolve the CSN paradox and suggest that the major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.

  11. Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways

    PubMed Central

    Kanika, Nirmala; Chang, Jinsook; Tong, Yuehong; Tiplitsky, Scott; Lin, Juan; Yohannes, Elizabeth; Tar, Moses; Chance, Mark; Christ, George J.; Melman, Arnold; Davies, Kelvin

    2010-01-01

    Objectives To investigate the role that oxidative stress plays in the development of diabetic cystopathy. Materials and methods Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month-old diabetic rats was carried out using microarray analysis. Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. The activity of protein degradation pathways was assessed using western blot analysis. Results Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10−10). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. Conclusions Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function. PMID:21518418

  12. A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins

    PubMed Central

    Sung, Min-Kyung; Porras-Yakushi, Tanya R; Reitsma, Justin M; Huber, Ferdinand M; Sweredoski, Michael J; Hoelz, André; Hess, Sonja; Deshaies, Raymond J

    2016-01-01

    Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19105.001 PMID:27552055

  13. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  14. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus.

    PubMed

    Shah, Firoz; Rineau, Francois; Canbäck, Björn; Johansson, Tomas; Tunlid, Anders

    2013-11-01

    Proteins contribute to a major part of the organic nitrogen (N) in forest soils. This N is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. The mechanisms by which these fungi depolymerize proteins and assimilate the released N are poorly characterized. Biochemical analysis and transcriptome profiling were performed to examine the proteolytic machinery and the uptake system of the ectomycorrhizal basidiomycete Paxillus involutus during the assimilation of organic N from various protein sources and extracts of organic matter. All substrates induced secretion of peptidase activity with an acidic pH optimum, mostly contributed by aspartic peptidases. The peptidase activity was transiently repressed by ammonium. Transcriptional analysis revealed a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. For the first time the molecular components of the protein degradation pathways of an ectomycorrhizal fungus are described. The data suggest that the transcripts encoding these components are regulated in response to the chemical properties and the availability of the protein substrates.

  15. Involvement of two latex-clearing proteins during rubber degradation and insights into the subsequent degradation pathway revealed by the genome sequence of Gordonia polyisoprenivorans strain VH2.

    PubMed

    Hiessl, Sebastian; Schuldes, Jörg; Thürmer, Andrea; Halbsguth, Tobias; Bröker, Daniel; Angelov, Angel; Liebl, Wolfgang; Daniel, Rolf; Steinbüchel, Alexander

    2012-04-01

    The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search.

  16. Involvement of Two Latex-Clearing Proteins during Rubber Degradation and Insights into the Subsequent Degradation Pathway Revealed by the Genome Sequence of Gordonia polyisoprenivorans Strain VH2

    PubMed Central

    Hiessl, Sebastian; Schuldes, Jörg; Thürmer, Andrea; Halbsguth, Tobias; Bröker, Daniel; Angelov, Angel; Liebl, Wolfgang; Daniel, Rolf

    2012-01-01

    The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search. PMID:22327575

  17. ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-04

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.

  18. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  19. EGF Uptake and Degradation Assay to Determine the Effect of HTLV Regulatory Proteins on the ESCRT-Dependent MVB Pathway.

    PubMed

    Murphy, Colin; Sheehy, Noreen

    2017-01-01

    The endosomal sorting complex required for transport (ESCRT) pathway plays key roles in multivesicular bodies (MVBs) formation and lysosomal degradation of membrane receptors, viral budding, and midbody abscission during cytokinesis. The epidermal growth factor receptor (EGFR) is regarded as a prototypical cargo of the MVB/ESCRT pathway and following stimulation by epidermal growth factor (EGF) EGFR/EGF complexes are internalized, sorted into MVBs, and degraded by lysosomes or recycled back to the cell membrane. Here, we describe an assay to analyze the effect of human T-cell leukemia (HTLV) regulatory proteins on the functionality of ESCRT-dependent MVB/lysosomal trafficking of EGFR/EGF complexes. This is performed by direct visualization and quantification of the rate of EGF-Alexa595/EGFR internalization and degradation in HeLa cells expressing HTLV regulatory proteins by immunofluorescence and western blot.

  20. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the curre...

  1. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-11-06

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

  2. Trafficking and degradation pathways in pathogenic conversion of prions and prion-like proteins in neurodegenerative diseases.

    PubMed

    Victoria, Guiliana Soraya; Zurzolo, Chiara

    2015-09-02

    Several neurodegenerative diseases such as transmissible spongiform encephalopathies, Alzheimer's and Parkinson's diseases are caused by the conversion of cellular proteins to a pathogenic conformer. Despite differences in the primary structure and subcellular localization of these proteins, which include the prion protein, α-synuclein and amyloid precursor protein (APP), striking similarity has been observed in their ability to seed and convert naïve protein molecules as well as transfer between cells. This review aims to cover what is known about the intracellular trafficking of these proteins as well as their degradation mechanisms and highlight similarities in their movement through the endocytic pathway that could contribute to the pathogenic conversion and seeding of these proteins which underlies the basis of these diseases.

  3. Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

    PubMed Central

    Kubota, Toru; Kita, Shunsuke; Nakatsu, Yuichiro; Aoki, Natsuko; Mori, Yoshio; Maenaka, Katsumi; Takeda, Makoto; Kidokoro, Minoru

    2014-01-01

    ABSTRACT Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection. PMID:25552722

  4. Immunity to killer toxin K1 is connected with the Golgi-to-vacuole protein degradation pathway.

    PubMed

    Valis, K; Masek, T; Novotná, D; Pospísek, M; Janderová, B

    2006-01-01

    Killer strains of Saccharomyces cerevisiae producing killer toxin K1 kill sensitive cells but are resistant to their own toxin. It is assumed that in the producer, an effective interaction between the external toxin and its plasma membrane receptor or the final effector is not possible on the grounds of a conformation change of the receptor or its absence in a membrane. Therefore, it is possible that some mutants with defects in intracellular protein transport and degradation can show a suicidal phenotype during K1 toxin production. We have examined these mutants in a collection of S. cerevisiae strains with deletions in various genes transformed by the pYX213+M1 vector carrying cDNA coding for the K1 toxin under the control of the GAL1 promoter. Determination of the quantity of dead cells in colony population showed that (1) the toxin production from the vector did not support full immunity of producing cells, (2) the suicidal phenotype was not connected with a defect in endocytosis or autophagy, (3) deletants in genes VPS1, VPS23, VPS51 and VAC8 required for the protein degradation pathway between the Golgi body and the vacuole exhibited the highest mortality. These results suggest that interacting molecule(s) on the plasma membrane in the producer might be diverted from the secretion pathway to degradation in the vacuole.

  5. Amyloid-Beta Protein Clearance and Degradation (ABCD) Pathways and their Role in Alzheimer’s Disease

    PubMed Central

    Baranello, Robert J.; Bharani, Krishna L.; Padmaraju, Vasudevaraju; Chopra, Nipun; Lahiri, Debomoy K.; Greig, Nigel H.; Pappolla, Miguel A.; Sambamurti, Kumar

    2016-01-01

    Amyloid-β proteins (Aβ) of 42 (Aβ42) and 40 aa (Aβ40) accumulate as senile plaques (SP) and cerebrovascular amyloid protein deposits that are defining diagnostic features of Alzheimer’s disease (AD). A number of rare mutations linked to familial AD (FAD) on the Aβ precursor protein (APP), Presenilin-1 (PS1), Presenilin-2 (PS2), Adamalysin10, and other genetic risk factors for sporadic AD such as the ε4 allele of Apolipoprotein E (ApoE-ε4) foster the accumulation of Aβ and also induce the entire spectrum of pathology associated with the disease. Aβ accumulation is therefore a key pathological event and a prime target for the prevention and treatment of AD. APP is sequentially processed by β-site APP cleaving enzyme (BACE1) and γ-secretase, a multisubunit PS1/PS2-containing integral membrane protease, to generate Aβ. Although Aβ accumulates in all forms of AD, the only pathways known to be affected in FAD increase Aβ production by APP gene duplication or via base substitutions on APP and γ-secretase subunits PS1 and PS2 that either specifically increase the yield of the longer Aβ42 or both Aβ40 and Aβ42. However, the vast majority of AD patients accumulate Aβ without these known mutations. This led to proposals that impairment of Aβ degradation or clearance may play a key role in AD pathogenesis. Several candidate enzymes, including Insulin-degrading enzyme (IDE), Neprilysin (NEP), Endothelin-converting enzyme (ECE), Angiotensin converting enzyme (ACE), Plasmin, and Matrix metalloproteinases (MMPs) have been identified and some have even been successfully evaluated in animal models. Several studies also have demonstrated the capacity of γ-secretase inhibitors to paradoxically increase the yield of Aβ and we have recently established that the mechanism is by skirting Aβ degradation. This review outlines major cellular pathways of Aβ degradation to provide a basis for future efforts to fully characterize the panel of pathways responsible for

  6. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways.

    PubMed

    Kerwin, Bruce A

    2008-08-01

    Polysorbates 20 and 80 (Tween 20 and Tween 80) are used in the formulation of biotherapeutic products for both preventing surface adsorption and as stabilizers against protein aggregation. The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan being polyoxyethylene sorbitan monolaurate for polysorbate 20 and polyoxyethylene sorbitan monooleate for polysorbate 80. The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate. This review focuses on the chemical structure of the polysorbates, factors influencing micelle formation and factors and excipients influencing stability and degradation of the polyoxyethylene and fatty acid ester linkages.

  7. Cellular senescence and protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Lessard, Frédéric; Gaumont-Leclerc, Marie-France; Bardeesy, Nabeel; Ferbeyre, Gerardo

    2014-01-01

    Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells. PMID:24866342

  8. Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug

    SciTech Connect

    Mosedale, Merrie; Wu, Hong; Kurtz, C. Lisa; Schmidt, Stephen P.; Adkins, Karissa; Harrill, Alison H.

    2014-10-01

    A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this drug. - Highlights: • Identified susceptible and resistant mouse strains to liver injury induced by a CAD • Liver injury characterized by single cell necrosis, and phospholipidosis

  9. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

    PubMed

    Dhar, Jayeeta; Barik, Sailen

    2016-12-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

  10. Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins

    PubMed Central

    Dhar, Jayeeta; Barik, Sailen

    2016-01-01

    Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar. PMID:27905537

  11. Experimental Priapism is Associated with Increased Oxidative Stress and Activation of Protein Degradation Pathways in Corporal Tissue

    PubMed Central

    Kanika, Nirmala D.; Melman, Arnold; Davies, Kelvin P.

    2010-01-01

    Priapism is a debilitating disease for which there is at present no clinically accepted pharmacologic intervention. It has been estimated that priapism lasting more than 24 hours in patients is associated with a 44–90% rate of erectile dysfunction (ED). In this investigation we determined in two animal models of priapism (opiorpin-induced priapism in the rat and priapism in a mouse model of sickle cell disease) if there is evidence for an increase in markers of oxidative stress in corporal tissue. In both animal models we demonstrate that priapism results in increased levels of lipid peroxidation, glutathione S-transferase activity, and oxidatively damaged proteins in corporal tissue. Using Western blot analysis we demonstrated there is up regulation of the ubiquitination ligase proteins, Nedd-4 and Mdm-2, and the lysososomal autophage protein, LC3. The anti-apoptotic protein, Bcl-2, was also up regulated. Overall, we demonstrate that priapism is associated with increased oxidative stress in corporal tissue and the activation of protein degradation pathways. Since oxidative stress is known to mediate the development of ED resulting from several etiologies (for example ED resulting from diabetes and aging) we suggest that damage to erectile tissue resulting from priapism might be prevented by treatments targeting oxidative stress. PMID:21085184

  12. Experimental priapism is associated with increased oxidative stress and activation of protein degradation pathways in corporal tissue.

    PubMed

    Kanika, N D; Melman, A; Davies, K P

    2010-01-01

    Priapism is a debilitating disease for which there is at present no clinically accepted pharmacological intervention. It has been estimated that priapism lasting more than 24 h in patients is associated with a 44-90% rate of ED. In this investigation, we determined in two animal models of priapism (opiorphin-induced priapism in the rat and priapism in a mouse model of sickle cell disease) if there is evidence for an increase in markers of oxidative stress in corporal tissue. In both animal models, we demonstrate that priapism results in increased levels of lipid peroxidation, glutathione S-transferase activity and oxidatively damaged proteins in corporal tissue. Using western blot analysis, we demonstrated there is upregulation of the ubiquitination ligase proteins, Nedd-4 and Mdm-2, and the lysosomal autophage protein, LC3. The antiapoptotic protein, Bcl-2, was also upregulated. Overall, we demonstrate that priapism is associated with increased oxidative stress in corporal tissue and the activation of protein degradation pathways. As oxidative stress is known to mediate the development of ED resulting from several etiologies (for example, ED resulting from diabetes and aging), we suggest that damage to erectile tissue resulting from priapism might be prevented by treatments targeting oxidative stress.

  13. Ubiquitin proteasome pathway-mediated degradation of proteins: effects due to site-specific substrate deamidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The accumulation, aggregation, and precipitation of proteins are etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome p...

  14. Interaction of structural core protein of Classical Swine Fever Virus with endoplasmic reticulum-associated degradation pathway protein OS9

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical Swine Fever Virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, t...

  15. Interaction of structural core protein of classical swine fever virus with endoplasmic reticulum-associated degradation pathway protein OS9.

    PubMed

    Gladue, D P; O'Donnell, V; Fernandez-Sainz, I J; Fletcher, P; Baker-Branstetter, R; Holinka, L G; Sanford, B; Carlson, J; Lu, Z; Borca, M V

    2014-07-01

    Classical swine fever virus (CSFV) Core protein is involved in virus RNA protection, transcription regulation and virus virulence. To discover additional Core protein functions a yeast two-hybrid system was used to identify host proteins that interact with Core. Among the identified host proteins, the osteosarcoma amplified 9 protein (OS9) was further studied. Using alanine scanning mutagenesis, the OS9 binding site in the CSFV Core protein was identified, between Core residues (90)IAIM(93), near a putative cleavage site. Truncated versions of Core were used to show that OS9 binds a polypeptide representing the 12 C-terminal Core residues. Cells transfected with a double-fluorescent labeled Core construct demonstrated that co-localization of OS9 and Core occurred only on unprocessed forms of Core protein. A recombinant CSFV containing Core protein where residues (90)IAIM(93) were substituted by alanines showed no altered virulence in swine, but a significant decreased ability to replicate in cell cultures.

  16. DHA inhibits protein degradation more efficiently than EPA by regulating the PPARγ/NFκB pathway in C2C12 myotubes.

    PubMed

    Wang, Yue; Lin, Qiao-wei; Zheng, Pei-pei; Zhang, Jian-song; Huang, Fei-ruo

    2013-01-01

    This study was conducted to evaluate the mechanism by which n-3 PUFA regulated the protein degradation in C2C12 myotubes. Compared with the BSA control, EPA at concentrations from 400 to 600 µM decreased total protein degradation (P < 0.01). However, the total protein degradation was decreased when the concentrations of DHA ranged from 300 µM to 700 µM (P < 0.01). DHA (400 µM, 24 h) more efficiently decreased the I κ B α phosphorylation and increased in the I κ B α protein level than 400 µM EPA (P < 0.01). Compared with BSA, 400 µM EPA and DHA resulted in a 47% or 68% induction of the NF κ B DNA binding activity, respectively (P < 0.01). Meanwhile, 400 µM EPA and DHA resulted in a 1.3-fold and 2.0-fold induction of the PPAR γ expression, respectively (P < 0.01). In C2C12 myotubes for PPAR γ knockdown, neither 400 µM EPA nor DHA affected the levels of p-I κ B α , total I κ B α or NF κ B DNA binding activity compared with BSA (P > 0.05). Interestingly, EPA and DHA both still decreased the total protein degradation, although PPAR γ knockdown attenuated the suppressive effects of EPA and DHA on the total protein degradation (P < 0.01). These results revealed that DHA inhibits protein degradation more efficiently than EPA by regulating the PPAR γ /NF- κ B pathway in C2C12 myotubes.

  17. Influence of oestradiol and tamoxifen on oestrogen receptors-alpha and -beta protein degradation and non-genomic signalling pathways in uterine and breast carcinoma cells.

    PubMed

    Horner-Glister, E; Maleki-Dizaji, M; Guerin, C J; Johnson, S M; Styles, J; White, I N H

    2005-12-01

    Tamoxifen acts as an oestrogen antagonist in the breast reducing cell proliferation, but in the uterus as an oestrogen agonist resulting in increased cell proliferation. Tamoxifen exerts its tissue-specific effects through the oestrogen receptors (ERalpha or ERbeta). The levels and functions of the two ERs affect the response of the target tissue to oestrogen and tamoxifen. We examined the control of ER stability in breast and uterine cell lines using western blotting and RT-PCR. In MCF-7 breast-derived cells, ERalpha and ERbeta proteins were rapidly degraded via the proteasome pathway in response to oestradiol; conversely tamoxifen stabilised both receptors. In Ishikawa uterine-derived cells, oestradiol and tamoxifen stabilised ERalpha but led to degradation of ERbeta by the proteasome pathway. Further investigations showed that oestradiol induced activation of the non-genomic ERalpha/Akt signalling pathway in MCF-7 cells. We have demonstrated that the alternative Erk signalling pathway is activated in Ishikawa cells following oestradiol treatment in the absence of an active proteasome pathway and therefore increased levels of ERbeta. In conclusion, our data have demonstrated tamoxifen or oestradiol control of ER subtype stability and that non-genomic activation of transcription pathways is cell specific.

  18. High mTORC1 signaling is maintained, while protein degradation pathways are perturbed in old murine skeletal muscles in the fasted state.

    PubMed

    White, Zoe; White, Robert B; McMahon, Christopher; Grounds, Miranda D; Shavlakadze, Tea

    2016-09-01

    This study investigated age-associated changes to protein synthesis and degradation pathways in the quadriceps muscles of male C57BL/6J mice at 5 ages, between 4 and 24 months (m). Sarcopenia was evident by 18m and was accompanied by hyper-phosphorylation of S6K1, indicating increased mTORC1 signaling. Proteasomal and autophagosomal degradation pathways were also impacted by aging. In the 1% NP40 insoluble protein fraction, the abundance of MuRF1 increased at 24m, while p62 increased at 15m, and remained elevated at older ages. In addition, we investigated how protein synthesis and degradation pathways are modulated by fasting in young (4m) and old (24m) muscles, and showed that old mice respond to fasting less robustly compared with young. Overnight fasting for 16h caused de-phosphorylation of AKT and molecules downstream of mTORC1 (S6K1, rpS6 and 4E-BP1) in young, but not old muscles. A longer time of fasting (24h) was required to reduce phosphorylation of these molecules in old mice. Induction of MuRF1 and Fbxo32 mRNA was also more robust in young compared with old muscles following fasting for 16h. In addition, a 16h fast reduced ULK1 phosphorylation at the mTORC1 specific site Ser757 only in young muscles. The striking accumulation of insoluble p62 protein in muscles of all old male mice (fed or fasted), suggests age-related dysregulation of autophagy and protein aggregation. These data provide an insight into the mechanisms of metabolic responses that affect protein homeostasis in old skeletal muscles, with applications to design of clinical interventions that target sarcopenia.

  19. A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana

    PubMed Central

    Li, Fangfang; Zhao, Nan; Xu, Xiongbiao; Wang, Yaqin; Yang, Xiuling; Liu, Shu-Sheng; Wang, Aiming; Zhou, Xueping

    2017-01-01

    A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway. PMID:28212430

  20. The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Binds to HIF-1α and Causes Its Degradation via the Proteasome Pathway

    PubMed Central

    Kousoulas, Konstantin G.

    2013-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761–6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway. PMID:23221556

  1. Short-term feed deprivation rapidly induces the protein degradation pathway in skeletal muscles of young mice.

    PubMed

    Shavlakadze, Tea; Soffe, Zoe; Anwari, Tahmina; Cozens, Greg; Grounds, Miranda D

    2013-04-01

    Analysis of protein kinase B (AKT) and S6 kinase1 (p70S6K) activity is widely used to assess the efficacy of interventions designed to increase or maintain skeletal muscle mass; these studies are often performed on feed-deprived mice. One problem associated with feed deprivation is that it promotes catabolism, and young or metabolically compromised mice may have less tolerance. The aim of our study was to determine the effect of various times of feed deprivation on the activity of AKT and p70S6K signaling and markers of protein catabolism in young, growing mice compared with adult mice. Young 23-d-old and adult 3-mo-old mice were feed deprived for 8, 10, and 12 h starting at 0700 h. In addition, adult mice were feed deprived for 24 h. AKT(Ser473) phosphorylation decreased by 50 and 76% from fed amounts by 10 and 12 h of feed deprivation, respectively, in young but not adult muscles. In adult muscles, feed deprivation for 24 h reduced AKT(Ser473) phosphorylation by 70%. Significant de-phosphorylation of p70S6K(Thr389) occurred in all feed-deprived young and adult mice. There was an increase in muscle RING-finger protein-1 (Murf1; 133-1245%) and muscle atrophy F-box protein or Atrogin-1 (Fbxo32; 210-2420%) mRNA in all young but not adult groups deprived of feed for 8-12 h, and there was a trend (P = 0.08) toward increased MURF1 associated with the contractile protein-enriched fraction isolated from young muscles of mice feed deprived for 12 h. This study demonstrates that skeletal muscles of young mice respond rapidly to feed deprivation by decreasing AKT activity and upregulating the protein degradation program.

  2. Differential Regulation of N-Myc and c-Myc Synthesis, Degradation, and Transcriptional Activity by the Ras/Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Kapeli, Katannya; Hurlin, Peter J.

    2011-01-01

    Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-RasG12V) in very different ways. H-RasG12V promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-RasG12V-mediated destabilization of N-Myc. We show that H-RasG12V-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis. PMID:21908617

  3. CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

    PubMed Central

    Schubert, Ulrich; Antón, Luis C.; Bačík, Igor; Cox, Josephine H.; Bour, Stéphane; Bennink, Jack R.; Orlowski, Marian; Strebel, Klaus; Yewdell, Jonathan W.

    1998-01-01

    ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked. PMID:9499087

  4. Inhibition of PCSK9 transcription by berberine involves down-regulation of hepatic HNF1α protein expression through the ubiquitin-proteasome degradation pathway.

    PubMed

    Dong, Bin; Li, Hai; Singh, Amar Bahadur; Cao, Aiqin; Liu, Jingwen

    2015-02-13

    Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α.

  5. Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.

    PubMed

    González, Alexis E; Muñoz, Vanessa C; Cavieres, Viviana A; Bustamante, Hianara A; Cornejo, Víctor-Hugo; Januário, Yunan C; González, Ibeth; Hetz, Claudio; daSilva, Luis L; Rojas-Fernández, Alejandro; Hay, Ronald T; Mardones, Gonzalo A; Burgos, Patricia V

    2017-03-02

    Brain regions affected by Alzheimer disease (AD) display well-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction. Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment β (C99), generated by cleavage of APP by β-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement in AD and the earliest initiator of synaptic plasticity and long-term memory impairment. The aim of the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosome formation or block the fusion of autophagosomes to endolysosomal compartments caused an increase in C99 levels. We also found that inhibition of autophagosome formation by depletion of Atg5 led to higher levels of C99 and to its massive accumulation in the lumen of enlarged perinuclear, lysosomal-associated membrane protein 1 (LAMP1)-positive organelles. In contrast, activation of autophagosome formation, either by starvation or by inhibition of the mammalian target of rapamycin, enhanced lysosomal clearance of C99. Altogether, our results indicate that autophagosomes are key organelles to help avoid C99 accumulation preventing its deleterious effects.-González, A. E., Muñoz, V. C., Cavieres, V. A., Bustamante, H. A., Cornejo, V.-H., Januário, Y. C., González, I., Hetz, C., daSilva, L. L., Rojas-Fernández, A., Hay, R. T., Mardones, G. A., Burgos, P. V. Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.

  6. Targeted protein degradation by PROTACs.

    PubMed

    Neklesa, Taavi K; Winkler, James D; Crews, Craig M

    2017-02-14

    Targeted protein degradation using the PROTAC technology is emerging as a novel therapeutic method to address diseases driven by the aberrant expression of a disease-causing protein. PROTAC molecules are bifunctional small molecules that simultaneously bind a target protein and an E3-ubiquitin ligase, thus causing ubiquitination and degradation of the target protein by the proteasome. Like small molecules, PROTAC molecules possess good tissue distribution and the ability to target intracellular proteins. Herein, we highlight the advantages of protein degradation using PROTACs, and provide specific examples where degradation offers therapeutic benefit over classical enzyme inhibition. Foremost, PROTACs can degrade proteins regardless of their function. This includes the currently "undruggable" proteome, which comprises approximately 85% of all human proteins. Other beneficial aspects of protein degradation include the ability to target overexpressed and mutated proteins, as well as the potential to demonstrate prolonged pharmacodynamics effect beyond drug exposure. Lastly, due to their catalytic nature and the pre-requisite ubiquitination step, an exquisitely potent molecules with a high degree of degradation selectivity can be designed. Impressive preclinical in vitro and in vivo PROTAC data have been published, and these data have propelled the development of clinically viable PROTACs. With the molecular weight falling in the 700-1000Da range, the delivery and bioavailability of PROTACs remain the largest hurdles on the way to the clinic. Solving these issues and demonstrating proof of concept clinical data will be the focus of many labs over the next few years.

  7. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  8. Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

    SciTech Connect

    Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung; Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho; Lee, Gye Won; Yun, Mi-Young; Cuong, Nguyen Manh; Shin, Jae-Gook; Song, Gyu-Yong; Oh, Sangtaek

    2010-01-01

    Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

  9. IL-1-induced ERK1/2 activation up-regulates p21{sup Waf1/Cip1} protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    SciTech Connect

    Arakawa, Tomohiro; Hayashi, Hidetoshi; Itoh, Saotomo; Takii, Takemasa; Onozaki, Kikuo

    2010-02-12

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21{sup Waf1/Cip1} (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  10. The Ski protein can inhibit ligand induced RARα and HDAC3 degradation in the Retinoic acid signaling pathway

    PubMed Central

    Zhao, Hongling; Ueki, Nobuhide; Marcelain, Katherine; Hayman, Michael J.

    2009-01-01

    Recent data has implicated the Ski protein as being a physiologically relevant negative regulator of signaling by Retinoic Acid (RA). The mechanism by which Ski represses RA signaling is unknown. Co-immunoprecipitation and immunofluorescence assay showed that Ski and RARα are in the same complex in both the absence and presence of RA, which makes Ski different from other corepressors. We determined that Ski can stabilize RARα and HDAC3. These results suggest that Ski represses RA signaling by stabilizing corepressor complex. PMID:19341714

  11. Photovoltaic lifetime and degradation science statistical pathway development: acrylic degradation

    NASA Astrophysics Data System (ADS)

    Bruckman, Laura S.; Wheeler, Nicholas R.; Kidd, Ian V.; Sun, Jiayang; French, Roger H.

    2013-09-01

    In order to optimize and extend the life of photovoltaics (PV) modules, scienti c and mechanistic statistical analytics must be performed on a large sample of materials, components and systems. Statistically signi - cant relationships were investigated between di erent mechanistically based variables to develop a statistical pathway diagram for the degradation of acrylic that is important in concentrating photovoltaics. The statisti- cally signi cant relationships were investigated using lifetime and degradation science using a domain knowledge semi-supervised generalized structural equation modeling (semi-gSEM. Predictive analytics and prognostics are informed from the statistical pathway diagram in order to predictively understand the lifetime of PV modules in di erent stress conditions and help with these critical lifetime technologies.

  12. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

    PubMed

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M; Zavala-Flores, Laura; Reyes-Reyes, Elsa M; Seravalli, Javier; Stanciu, Lia A; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2015-09-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

  13. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways

    PubMed Central

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M.; Zavala-Flores, Laura; Reyes-Reyes, Elsa M.; Seravalli, Javier; Stanciu, Lia A.; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2014-01-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson’s disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of WT or A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic cells and yeast in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways. PMID:25497688

  14. Rat skeletal muscle glycogen degradation pathways reveal differential association of glycogen-related proteins with glycogen granules.

    PubMed

    Xu, Hongyang; Stapleton, David; Murphy, Robyn M

    2015-06-01

    Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzymes that contribute to glycogen particle metabolism. In Long-Evans Hooded rat whole muscle homogenates prepared from extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, oxidative, predominantly slow twitch), it was necessary to include α-amylase, which releases glucosyl units from glycogen, to detect glycogenin but not GDE or GP. Up to ∼12 % of intramuscular glycogen pool was broken down using either in vitro electrical stimulation or leaving muscle at room temperature >3 h (delayed, post-mortem). Electrical stimulation did not reveal glycogenin unless α-amylase was added, although in post-mortem muscle ∼50 and ∼30 % of glycogenin in EDL and SOL muscles, respectively, was detected compared to the amount detected with α-amylase treatment. Single muscle fibres were dissected from fresh or post-mortem EDL muscles, mechanically skinned to remove surface membrane and the presence of glycogenin, GDE and GP as freely diffusible proteins (i.e. cytoplasmic localization) compared by Western blotting. Diffusibility of glycogenin (∼20 %) and GP (∼60 %) was not different between muscles, although GDE increased from ∼15 % diffusible in fresh muscle to ∼60 % in post-mortem muscle. Under physiologically relevant circumstances, in rat muscle and within detection limits: (1) The total cellular pool of glycogenin is always associated with glycogen granules, (2) GDE is associated with glycogen granules with over half the total pool associated with the outer tiers of glycogen, (3) GP is only ever weakly associated with glycogen granules and (4) addition of α-amylase is necessary in order to detect glycogenin, but not GDE or GP.

  15. A d-Amino Acid at the N-Terminus of a Protein Abrogates Its Degradation by the N-End Rule Pathway

    PubMed Central

    2015-01-01

    Eukaryotes have evolved the ubiquitin (Ub)/proteasome system to degrade polypeptides. The Ub/proteasome system is one way that cells regulate cytosolic protein and amino acids levels through the recognition and ubiquitination of a protein’s N-terminus via E1, E2, and E3 enzymes. The process by which the N-terminus stimulates intracellular protein degradation is referred to as the N-end rule. Characterization of the N-end rule has been limited to only the natural l-amino acids. Using a cytosolic delivery platform derived from anthrax lethal toxin, we probed the stability of mixed chirality proteins, containing one d-amino acid on the N-terminus of otherwise all l-proteins. In all cases, we observed that one N-terminal d-amino acid stabilized the cargo protein to proteasomal degradation with respect to the N-end rule. We found that since the mixed chirality proteins were not polyubiquitinated, they evaded N-end-mediated proteasomal degradation. Evidently, a subtle change on the N-terminus of a natural protein can enhance its intracellular lifetime. PMID:26807441

  16. Fos family protein degradation by the proteasome.

    PubMed

    Gomard, Tiphanie; Jariel-Encontre, Isabelle; Basbous, Jihane; Bossis, Guillaume; Moquet-Torcy, Gabriel; Mocquet-Torcy, Gabriel; Piechaczyk, Marc

    2008-10-01

    c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.

  17. BACE2 degradation mediated by the macroautophagy-lysosome pathway.

    PubMed

    Liu, Xi; Wang, Zhe; Wu, Yili; Wang, Jianping; Song, Weihong

    2013-06-01

    Neuritic plaque is the pathological hallmark in Alzheimer's disease (AD). Amyloid-β protein (Aβ), the central component of neuritic plaques, is generated from amyloid-β precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. β-site APP cleaving enzyme 2 (BACE2), a homolog of BACE1, functions differently from BACE1 in APP processing. BACE1 is the β-secretase essential for Aβ production, and BACE2, a θ-secretase, cleaves APP within the Aβ domain, preventing Aβ production. Elucidation of the mechanism underlying BACE2 degradation is important for defining its biological features and its potential role in Alzheimer's disease drug development. In this report we first showed that the half-life of BACE2 is approximately 20 h. Lysosomal inhibition increased BACE2 protein levels whereas proteasomal inhibition had no effect on BACE2 protein expression. Furthermore, we identified that macroautophagy mediated BACE2 degradation. Finally, we showed that lysosomal inhibition increased BACE2 cleavage of APP. Taken together, our in vitro study showed that BACE2 is degraded through the macrophagy-lysosome pathway and that lysosomal inhibition affects BACE2 processing of APP. Modulation of BACE2 degradation via the lysosomal pathway could be a new target for AD drug development.

  18. Global subcellular characterization of protein degradation using quantitative proteomics.

    PubMed

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J; Ly, Tony; Lamond, Angus I

    2013-03-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ~5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.

  19. Hyperglycemia triggers HIPK2 protein degradation

    PubMed Central

    Granato, Marisa; Cuomo, Laura; Pistritto, Giuseppa; Cirone, Mara; D'Orazi, Gabriella

    2017-01-01

    Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies. PMID:27901482

  20. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    PubMed Central

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  1. SNIPER peptide-mediated degradation of endogenous proteins.

    PubMed

    Fan, Xuelai; Wang, Yu Tian

    2015-03-02

    Rapid and reversible methods for altering the function of endogenous proteins are not only indispensable tools for probing complex biological systems, but may potentially drive the development of new therapeutics for the treatment of human diseases. Genetic approaches have provided insights into protein function, but are limited in speed, reversibility and spatiotemporal control. To overcome these limitations, we have developed a peptide-based method (SNIPER: Selective Native Protein Eradication) to degrade any given endogenous protein at the post-translational level by harnessing chaperone-mediated autophagy, a major intracellular protein degradation pathway. This unit presents a typical strategy in the design and validation of a protein-knockdown peptide.

  2. Foot-and-mouth disease virus structural protein VP3 degrades Janus kinase 1 to inhibit IFN-γ signal transduction pathways

    PubMed Central

    Li, Dan; Wei, Jin; Yang, Fan; Liu, Hua-Nan; Zhu, Zi-Xiang; Cao, Wei-Jun; Li, Shu; Liu, Xiang-Tao; Zheng, Hai-Xue; Shu, Hong-Bing

    2016-01-01

    ABSTRACT Foot-and-mouth disease is a highly contagious viral disease of cloven-hoofed animals that is caused by foot-and-mouth disease virus (FMDV). To replicate efficiently in vivo, FMDV has evolved methods to circumvent host antiviral defense mechanisms, including those induced by interferons (IFNs). Previous research has focused on the effect of FMDV Lpro and 3Cpro on type I IFNs. In this study, FMDV VP3 was found to inhibit type II IFN signaling pathways. The overexpression of FMDV VP3 inhibited the IFN-γ-triggered phosphorylation of STAT1 at Tyr701 and the subsequent expression of downstream genes. Mechanistically, FMDV VP3 interacted with JAK1/2 and inhibited the tyrosine phosphorylation, dimerization and nuclear accumulation of STAT1. FMDV VP3 also disrupted the assembly of the JAK1 complex and degraded JAK1 but not JAK2 via a lysosomal pathway. Taken together, the results reveal a novel mechanism used by which FMDV VP3 counteracts the type II IFN signaling pathways. PMID:26901336

  3. PATHWAYS - ELECTRON TUNNELING PATHWAYS IN PROTEINS

    NASA Technical Reports Server (NTRS)

    Beratan, D. N.

    1994-01-01

    The key to understanding the mechanisms of many important biological processes such as photosynthesis and respiration is a better understanding of the electron transfer processes which take place between metal atoms (and other groups) fixed within large protein molecules. Research is currently focused on the rate of electron transfer and the factors that influence it, such as protein composition and the distance between metal atoms. Current models explain the swift transfer of electrons over considerable distances by postulating bridge-mediated tunneling, or physical tunneling pathways, made up of interacting bonds in the medium around and between donor and acceptor sites. The program PATHWAYS is designed to predict the route along which electrons travel in the transfer processes. The basic strategy of PATHWAYS is to begin by recording each possible path element on a connectivity list, including in each entry which two atoms are connected and what contribution the connection would make to the overall rate if it were included in a pathway. The list begins with the bonded molecular structure (including the backbone sequence and side chain connectivity), and then adds probable hydrogen bond links and through-space contacts. Once this list is completed, the program runs a tree search from the donor to the acceptor site to find the dominant pathways. The speed and efficiency of the computer search offers an improvement over manual techniques. PATHWAYS is written in FORTRAN 77 for execution on DEC VAX series computers running VMS. The program inputs data from four data sets and one structure file. The software was written to input BIOGRAF (old format) structure files based on x-ray crystal structures and outputs ASCII files listing the best pathways and BIOGRAF vector files containing the paths. Relatively minor changes could be made in the input format statements for compatibility with other graphics software. The executable and source code are included with the

  4. Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

    PubMed Central

    Tropel, David; van der Meer, Jan Roelof

    2004-01-01

    Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways. PMID:15353566

  5. A superoxide-mediated mitogen-activated protein kinase phosphatase-1 degradation and c-Jun NH(2)-terminal kinase activation pathway for luteolin-induced lung cancer cytotoxicity.

    PubMed

    Bai, Lang; Xu, Xiuling; Wang, Qiong; Xu, Shanling; Ju, Wei; Wang, Xia; Chen, Wenshu; He, Weiyang; Tang, Hong; Lin, Yong

    2012-04-01

    Although luteolin is identified as a potential cancer therapeutic and preventive agent because of its potent cancer cell-killing activity, the molecular mechanisms by which its cancer cell cytotoxicity is achieved have not been well elucidated. In this report, luteolin-induced cellular signaling was systematically investigated, and a novel pathway for luteolin's lung cancer killing was identified. The results show that induction of superoxide is an early and crucial step for luteolin-induced apoptotic and nonapoptotic death in lung cancer cells. The c-Jun N-terminal kinase (JNK) was potently activated after superoxide accumulation. Suppression of superoxide completely blocked luteolin-induced JNK activation, which was well correlated to alleviation of luteolin's cytotoxicity. Although luteolin slightly stimulated the JNK-activating kinase mitogen-activated protein kinase kinase 7, the latter was not dependent on superoxide. We further found that luteolin triggers a superoxide-dependent rapid degradation of the JNK-inactivating phosphatase mitogen-activated protein kinase phosphatase-1 (MKP-1). Introduction of a degradation-resistant MKP-1 mutant effectively attenuated luteolin-induced JNK activation and cytotoxicity, suggesting that inhibition of the JNK suppressor MKP-1 plays a major role in luteolin-induced lung cancer cell death. Taken together, our results unveil a novel pathway consisting of superoxide, MKP-1, and JNK for luteolin's cytotoxicity in lung cancer cells, and manipulation of this pathway could be a useful approach for applying luteolin for lung cancer prevention and therapy.

  6. Protein design for pathway engineering

    SciTech Connect

    Eriksen, DT; Lian, JZ; Zhao, HM

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

  7. Protein Design for Pathway Engineering

    PubMed Central

    Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

    2013-01-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

  8. Protein design for pathway engineering.

    PubMed

    Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.

  9. Salidroside stimulates the accumulation of HIF-1α protein resulted in the induction of EPO expression: a signaling via blocking the degradation pathway in kidney and liver cells.

    PubMed

    Zheng, Ken Yu-Zhong; Zhang, Zhen-Xia; Guo, Ava Jiang-Yang; Bi, Cathy Wen-Chuang; Zhu, Kevin Yue; Xu, Sherry Li; Zhan, Janis Ya-Xian; Lau, David Tai-Wei; Dong, Tina Ting-Xia; Choi, Roy Chi-Yan; Tsim, Karl Wah-Keung

    2012-03-15

    Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance to mountain sickness in preventing hypoxia; however, the functional ingredient responsible for this adaptogenic effect has not been revealed. Here, we have identified salidroside, a glycoside predominantly found in Rhodiola, is the chemical in providing such anti-hypoxia effect. Cultured human embryonic kidney fibroblast (HEK293T) and human hepatocellular carcinoma (HepG2) were used to reveal the mechanism of this hematopoietic function mediated by salidroside. The application of salidroside in cultures induced the expression of erythropoietin (EPO) mRNA from its transcription regulatory element hypoxia response element (HRE), located on EPO gene. The application of salidroside stimulated the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein, but not HIF-2α protein: the salidroside-induced HIF-1α protein was via the reduction of HIF-1α degradation but not the mRNA induction. The increased HIF-1α could account for the activation of EPO gene. These results supported the notion that hematopoietic function of Rhodiola was triggered, at least partially, by salidroside.

  10. Metabolic Pathways for Degradation of Aromatic Hydrocarbons by Bacteria.

    PubMed

    Ladino-Orjuela, Guillermo; Gomes, Eleni; da Silva, Roberto; Salt, Christopher; Parsons, John R

    2016-01-01

    The aim of this review was to build an updated collection of information focused on the mechanisms and elements involved in metabolic pathways of aromatic hydrocarbons by bacteria. Enzymes as an expression of the genetic load and the type of electron acceptor available, as an environmental factor, were highlighted. In general, the review showed that both aerobic routes and anaerobic routes for the degradation of aromatic hydrocarbons are divided into two pathways. The first, named the upper pathways, entails the route from the original compound to central intermediate compounds still containing the aromatic ring but with the benzene nucleus chemically destabilized. The second, named the lower pathway, begins with ring de-aromatization and subsequent cleavage, resulting in metabolites that can be used by bacteria in the production of biomass. Under anaerobic conditions the five mechanisms of activation of the benzene ring described show the diversity of chemical reactions that can take place. Obtaining carbon and energy from an aromatic hydrocarbon molecule is a process that exhibits the high complexity level of the metabolic apparatus of anaerobic microorganisms. The ability of these bacteria to express enzymes that catalyze reactions, known only in non-biological conditions, using final electron acceptors with a low redox potential, is a most interesting topic. The discovery of phylogenetic and functional characteristics of cultivable and noncultivable hydrocarbon degrading bacteria has been made possible by improvements in molecular research techniques such as SIP (stable isotope probing) tracing the incorporation of (13)C, (15)N and (18)O into nucleic acids and proteins. Since many metabolic pathways in which enzyme and metabolite participants are still unknown, much new research is required. Therefore, it will surely allow enhancing the known and future applications in practice.

  11. Protein Degradation and the Stress Response

    PubMed Central

    Flick, Karin; Kaiser, Peter

    2012-01-01

    Environmental stresses are manifold and so are the responses they elicit. This is particularly true for higher eukaryotes where various tissues and cell types are differentially affected by the insult. Type and scope of the stress response can therefore differ greatly among cell types. Given the importance of the Ubiquitin Proteasome System (UPS) for most cellular processes, it comes as no surprise that the UPR plays a pivotal role in counteracting the effects of stressors. Here we outline contributions of the UPS to stress sensing, signaling, and response pathways. We make no claim to comprehensiveness but choose selected examples to illustrate concepts and mechanisms by which protein modification with ubiquitin and proteasomal degradation of key regulators ensures cellular integrity during stress situations. PMID:22414377

  12. Protein oxidation and degradation caused by particulate matter

    PubMed Central

    Lai, Ching-Huang; Lee, Chun-Nin; Bai, Kuan-Jen; Yang, You-Lan; Chuang, Kai-Jen; Wu, Sheng-Ming; Chuang, Hsiao-Chi

    2016-01-01

    Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black (CB; control particles). Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM. PMID:27644844

  13. Protein oxidation and degradation caused by particulate matter

    NASA Astrophysics Data System (ADS)

    Lai, Ching-Huang; Lee, Chun-Nin; Bai, Kuan-Jen; Yang, You-Lan; Chuang, Kai-Jen; Wu, Sheng-Ming; Chuang, Hsiao-Chi

    2016-09-01

    Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black (CB; control particles). Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM.

  14. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  15. Ubiquitin-mediated protein degradation in Xenopus egg extracts.

    PubMed

    Castro, Anna; Vigneron, Suzanne; Bernis, Cyril; Labbé, Jean-Claude; Lorca, Thierry

    2006-01-01

    Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation. Xenopus egg extracts present many advantages for the study of the cell cycle, including the availability of a large quantity of material synchronized at a particular phase of the cell cycle. In this chapter, we describe various protocols used in Xenopus egg extracts to study the ubiquitination and degradation of different cell cycle regulators. We first provide the method used to obtain interphase- and metaphase II-arrested egg extracts. Subsequently, we describe the protocol employed in these extracts to test the putative ubiquitination and degradation of a protein. Moreover, we describe a detailed practical procedure to test the role of different regulators in the ubiquitin-dependent degradation pathway of a specific protein. To that, we show how to eliminate some of these regulators from the extracts by immunodepletion and how to activate ectopically their function by the translation of their messenger ribonucleic acid. Finally, the Notes provide a series of practical details that explain the different problems that can occur and the possible solutions used to overcome them.

  16. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  17. Enhanced Degradation of Misfolded Proteins Promotes Tumorigenesis.

    PubMed

    Chen, Liang; Brewer, Michael D; Guo, Lili; Wang, Ruoxing; Jiang, Peng; Yang, Xiaolu

    2017-03-28

    An adequate cellular capacity to degrade misfolded proteins is critical for cell survival and organismal health. A diminished capacity is associated with aging and neurodegenerative diseases; however, the consequences of an enhanced capacity remain undefined. Here, we report that the ability to clear misfolded proteins is increased during oncogenic transformation and is reduced upon tumor cell differentiation. The augmented capacity mitigates oxidative stress associated with oncogenic growth and is required for both the initiation and maintenance of malignant phenotypes. We show that tripartite motif-containing (TRIM) proteins select misfolded proteins for proteasomal degradation. The higher degradation power in tumor cells is attributed to the upregulation of the proteasome and especially TRIM proteins, both mediated by the antioxidant transcription factor Nrf2. These findings establish a critical role of TRIMs in protein quality control, connect the clearance of misfolded proteins to antioxidant defense, and suggest an intrinsic characteristic of tumor cells.

  18. The M2 module of the Cys-His-rich domain (CHRD) of PCSK9 protein is needed for the extracellular low-density lipoprotein receptor (LDLR) degradation pathway.

    PubMed

    Saavedra, Yascara Grisel Luna; Day, Robert; Seidah, Nabil G

    2012-12-21

    PCSK9 enhances the cellular degradation of the LDL receptor (LDLR), leading to increased plasma LDL cholesterol. This multidomain protein contains a prosegment, a catalytic domain, a hinge region, and a cysteine-histidine rich domain (CHRD) composed of three tightly packed modules named M1, M2, and M3. The CHRD is required for the activity of PCSK9, but the mechanism behind this remains obscure. To define the contribution of each module to the function of PCSK9, we dissected the CHRD structure. Six PCSK9 deletants were generated by mutagenesis, corresponding to the deletion of only one (ΔM1, ΔM2, ΔM3) or two (ΔM12, ΔM13, ΔM23) modules. Transfection of HEK293 cells showed that all deletants were well processed and expressed compared with the parent PCSK9 but that only those lacking the M2 module were secreted. HepG2 cells lacking endogenous PCSK9 (HepG2/shPCSK9) were used for the functional analysis of the extracellular or intracellular activity of PCSK9 and its deletants. To analyze the ability of the deletants to enhance the LDLR degradation by the intracellular pathway, cellular expressions revealed that only the ΔM2 deletant retains a comparable total LDLR-degrading activity to full-length PCSK9. To probe the extracellular pathway, HepG2/shPCSK9 cells were incubated with conditioned media from transfected HEK293 or HepG2/shPCSK9 cells, and cell surface LDLR levels were analyzed by FACS. The results showed no activity of any secreted deletant compared with PCSK9. Thus, although M2 is dispensable for secretion, its presence is required for the extracellular activity of PCSK9 on cell surface LDLR.

  19. Understanding Degradation Pathways in Organic Photovoltaics (Poster)

    SciTech Connect

    Lloyd, M. T.; Olson, D. C.; Garcia, A.; Kauvar, I.; Kopidakis, N.; Reese, M. O.; Berry, J. J.; Ginley, D. S.

    2011-02-01

    Organic Photovoltaics (OPVs) recently attained power conversion efficiencies that are of interest for commercial production. Consequently, one of the most important unsolved issues facing a new industry is understanding what governs lifetime in organic devices and discovering solutions to mitigate degradation mechanisms. Historically, the active organic components are considered vulnerable to photo-oxidation and represent the primary degradation channel. However, we present several (shelf life and light soaking) studies pointing the relative stability of the active layers and instabilities in commonly used electrode materials. We show that engineering of the hole/electron layer at the electrode can lead to environmentally stable devices even without encapsulation.

  20. Protein quality control, retention, and degradation at the endoplasmic reticulum.

    PubMed

    Benyair, Ron; Ron, Efrat; Lederkremer, Gerardo Z

    2011-01-01

    In order to maintain proper cellular functions, all living cells, from bacteria to mammalian cells, must carry out a rigorous quality control process in which nascent and newly synthesized proteins are examined. An important role of this process is to protect cells against pathological accumulation of unfolded and misfolded proteins. The endoplasmic reticulum (ER) has evolved as a staging ground for secretory protein synthesis with distinct sites for entry, quality control, and exit. In the ER, most proteins are N-glycosylated, a posttranslational modification that defines the quality control pathway that the protein will undergo. The folding state of glycoproteins is revealed by specific modifications of their N-glycans. Regardless of size and posttranslational modifications, the folding states of all proteins must be identified as unfolded, properly folded, or terminally misfolded and accordingly subjected to ER retention and continued folding attempts, export and maturation, or retrotranslocation to the cytosol for degradation. These processes involve specialized machineries that utilize molecular chaperones, protein- and N-glycan-modifying enzymes, and lectins for protein folding and quality control and ubiquitination and degradation machineries for disposal. All these machineries are regulated by a signaling pathway, the unfolded protein response, which upregulates ER functions when under the stress of high protein load. Here, we describe the molecular mechanisms that are implicated and discuss recent data that underline the importance of compartmentalization in the segregation of the various functions of the ER for their correct function.

  1. Degradation-mediated protein quality control at the inner nuclear membrane

    PubMed Central

    Boban, Mirta; Foisner, Roland

    2016-01-01

    abstract An intricate machinery protects cells from the accumulation of misfolded, non-functional proteins and protein aggregates. Protein quality control pathways have been best described in the cytoplasm and the endoplasmic reticulum, however, recent findings indicate that the nucleus is also an important compartment for protein quality control. Several nuclear ubiquitinylation pathways target soluble and membrane proteins in the nucleus and mediate their degradation through nuclear proteasomes. In addition, emerging data suggest that nuclear envelope components are also degraded by autophagy, although the mechanisms by which cytoplasmic autophagy machineries get access to nuclear targets remain unclear. In this minireview we summarize the nuclear ubiquitin-proteasome pathways in yeast, focusing on pathways involved in the protein degradation at the inner nuclear membrane. In addition, we discuss potential mechanisms how nuclear targets at the nuclear envelope may be delivered to the cytoplasmic autophagy pathways in yeast and mammals. PMID:26760377

  2. Targeted Protein Degradation by Small Molecules.

    PubMed

    Bondeson, Daniel P; Crews, Craig M

    2017-01-06

    Protein homeostasis networks are highly regulated systems responsible for maintaining the health and productivity of cells. Whereas therapeutics have been developed to disrupt protein homeostasis, more recently identified techniques have been used to repurpose homeostatic networks to effect degradation of disease-relevant proteins. Here, we review recent advances in the use of small molecules to degrade proteins in a selective manner. First, we highlight all-small-molecule techniques with direct clinical application. Second, we describe techniques that may find broader acceptance in the biomedical research community that require little or no synthetic chemistry. In addition to serving as innovative research tools, these new approaches to control intracellular protein levels offer the potential to develop novel therapeutics targeting proteins that are not currently pharmaceutically vulnerable.

  3. Degradable PEGylated Protein Conjugates Utilizing RAFT Polymerization.

    PubMed

    Decker, Caitlin G; Maynard, Heather D

    2015-04-01

    Poly(ethylene glycol) (PEG)-protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body. Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone. Herein we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn ) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Ð) of 1.34 and 1.71, respectively. Hydrolytic degradation of the polymers was analyzed by (1)H-NMR and GPC under basic and acidic conditions. The reversible covalent attachment of these polymers to lysozyme, as well as the hydrolytic and reductive cleavage of the polymer from the protein, was analyzed by gel electrophoresis and mass spectrometry. Following reductive cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity. This represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable.

  4. Degradation of Akt Using Protein Catalyzed Capture Agents

    PubMed Central

    Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M.; Heath, James R.

    2016-01-01

    Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry we recently developed multiple protein catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein based assay. Here we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare Proteolysis Targeting Chimeric molecules (PROTACs) that are shown to promote the rapid degradation of Akt in live cancer cells. These novel PROTACs demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. PMID:26880702

  5. Cathodic degradation of antibiotics: characterization and pathway analysis.

    PubMed

    Kong, Deyong; Liang, Bin; Yun, Hui; Cheng, Haoyi; Ma, Jincai; Cui, Minhua; Wang, Aijie; Ren, Nanqi

    2015-04-01

    Antibiotics in wastewaters must be degraded to eliminate their antibacterial activity before discharging into the environment. A cathode can provide continuous electrons for the degradation of refractory pollutants, however the cathodic degradation feasibility, efficiency and pathway for different kinds of antibiotics is poorly understood. Here, we investigated the degradation of four antibiotics, namely nitrofurazone (NFZ), metronidazole (MNZ), chloramphenicol (CAP), and florfenicol (FLO) by a poised cathode in a dual chamber electrochemical reactor. The cyclic voltammetry preliminarily proved the feasibility of the cathodic degradation of these antibiotics. The cathodic reducibility of these antibiotics followed the order of NFZ > MNZ > CAP > FLO. A decreased phosphate buffered solution (PBS) concentration as low as 2 mM or utilization of NaCl buffer solution as catholyte had significant influence on antibiotics degradation rate and efficiency for CAP and FLO but not for NFZ and MNZ. PBS could be replaced by Na2CO3-NaHCO3 buffer solution as catholyte for the degradation of these antibiotics. Reductive dechlorination of CAP proceeded only after the reduction of the nitro group to aromatic amine. The composition of the degradation products depended on the cathode potential except for MNZ. The cathodic degradation process could eliminate the antibacterial activity of these antibiotics. The current study suggests that the electrochemical reduction could serve as a potential pretreatment or advanced treatment unit for the treatment of antibiotics containing wastewaters.

  6. A new microbial degradation pathway of steroid alkaloids.

    PubMed

    Gaberc-Porekar, V; Gottlieb, H E; Mervic, M

    1983-10-01

    In the degradation pathway of the steroid alkaloid tomatidine by Gymnoascus reesii the A-ring of tomatidine is opened with the formation of the 4-hydroxy-3,4-secotomatidine-3-oic acid, which was identified in the form of N-acetyl-3,4-tomatidine-carbolactone by mass, IR and 1H NMR spectra. Cleavage of the A-ring in the starting reaction indicates that an alternative pathway must be operating, instead of the general oxidative one.

  7. Hydroxide Degradation Pathways for Imidazolium Cations. A DFT Study

    SciTech Connect

    Long, H.; Pivovar, B.

    2014-05-15

    Imidazolium cations are promising candidates as covalently tetherable cations for application in anion exchange membranes. They have generated specific interest in alkaline membrane fuel cell applications where ammonium-based cations have been the most commonly applied but have been found to be susceptible to hydroxide attack. In the search for high stability cations, a detailed understanding of the degradation pathways and reaction barriers is required. In this work, we investigate imidazolium and benzimidazolium cations in the presence of hydroxide using density functional theory calculations for their potential in alkaline membrane fuel cells. Moreover, the dominant degradation pathway for these cations is predicted to be the nucleophilic addition–elimination pathway at the C-2 atom position on the imidazolium ring. Steric interferences, introduced by substitutions at the C-2, C-4, and C-5 atom positions, were investigated and found to have a significant, positive impact on calculated degradation energy barriers. Benzimidazolium cations, with their larger conjugated systems, are predicted to degrade much faster than their imidazolium counterparts. Our results provide important insight into designing stable cations for anion exchange membranes. Some of the molecules studied have significantly increased degradation energy barriers suggesting that they could possess significantly improved (several orders of magnitude) durability compared to traditional cations and potentially enable new applications.

  8. ORGANOPHOSPHATE PESTICIDE DEGRADATION PATHWAYS DURING DRINKING WATER TREATMENT

    EPA Science Inventory

    Free chlorine has been found to react with organophosphate (OP) pesticides resulting in the more toxic oxon products. We will discuss OP pesticide degradation pathways and modeling in the presence of chlorine and chloramines, as well as present a relationship between structure a...

  9. Selective Target Protein Degradation via Phthalimide Conjugation

    PubMed Central

    Winter, Georg E.; Buckley, Dennis L.; Paulk, Joshiawa; Roberts, Justin M.; Souza, Amanda; Dhe-Paganon, Sirano; Bradner, James E.

    2016-01-01

    Small-molecule antagonists disable discrete biochemical properties of protein targets. For multi-domain protein targets, the pharmacologic consequence of drug action is limited by selective disruption of one domain-specific activity. More broadly, target inhibition is kinetically limited by the durability and degree of target engagement. These features of traditional drug molecules are challenging to the development of inhibitors targeting transcription factors and chromatin-associated epigenetic proteins, which function as multi-domain biomolecular scaffolds and generally feature rapid association and dissociation kinetics. We therefore devised a chemical strategy to prompt ligand-dependent target protein degradation, via chemical conjugation with derivatized phthalimides that hijack the function of the Cereblon E3 ubiquitin ligase complex. Using this approach, we converted an acetyl-lysine competitive antagonist that displaces BET bromodomains from chromatin (JQ1) to a phthalimide-conjugated ligand that prompts immediate Cereblon-dependent BET protein degradation (dBET1). Expression proteomics confirms high specificity for BET family members BRD2, BRD3 and BRD4 among 7429 proteins detected. Degradation of BET bromodomains is associated with a more rapid and robust apoptotic response compared to bromodomain inhibition in primary human leukemic blasts, and dBET1 exhibits in vivo efficacy in a human leukemia xenograft. The reach of this approach is illustrated by a second series of probes that degrade the cytosolic signaling protein, FKBP12. Together, these findings identify a facile and general new strategy to control target protein stability, with implications for approaching previously intractable protein targets. PMID:25999370

  10. Pathways of 4-hydroxybenzoate degradation among species of Bacillus.

    PubMed

    Crawford, R L

    1976-07-01

    The pathways used by three bacterial strains of the genus Bacillus to degrade 4-hydroxybenzoate are delineated. When B. brevis strain PHB-2 is grown on 4-hydroxybenzoate, enzymes of the protocatechuate branch of the beta-ketoadipate pathway are induced. In contrast, B. circulans strain 3 contains high levels of the enzymes of the protocatechuate 2,3-dioxygenase pathway after growth on 4-hydroxybenzoate. B. laterosporus strain PHB-7a degrades 4-hydroxybenzoate by a novel reaction sequence. After growth on 4-hydroxybenzoate, strain PHB-7a contains high levels of gentisate oxygenase (EC 1.13.11.4) and maleylpyruvate hydrolase. Whole cells of strain PHB-7a (grown on 4-hydroxylbenzoate) accumulate 2,5-dihydroxybenzoate (gentisate) from 4-hydroxybenzoate when incubated in the presence of 1mM alpha,alpha'-dipyridyl. Thus, strain PHB-7a appears to convert 4-hydroxybenzoate to gentisate, which is further degraded by the glutathione-independent gentisic acid pathway. These pathway delineations provide evidence that Bacillus species are derived from a diverse evolutionary background.

  11. A permeabilized cell system identifies the endoplasmic reticulum as a site of protein degradation

    PubMed Central

    1991-01-01

    Analysis of the fate of a variety of newly synthesized proteins in the secretory pathway has provided evidence for the existence of a novel protein degradation system distinct from that of the lysosome. Although current evidence suggests that proteins degraded by this system are localized to a pre-Golgi compartment before degradation, the site of proteolysis has not been determined. A permeabilized cell system was developed to examine whether degradation by this pathway required transport out of the ER, and to define the biochemical characteristics of this process. Studies were performed on fibroblast cell lines expressing proteins known to be sensitive substrates for this degradative process, such as the chimeric integral membrane proteins, Tac-TCR alpha and Tac-TCR beta. By immunofluorescence microscopy, these proteins were found to be localized to the ER. Treatment with cycloheximide resulted in the progressive disappearance of intracellular staining without change in the ER localization of the chimeric proteins. Cells permeabilized with the pore-forming toxin streptolysin O were able to degrade these newly synthesized proteins. The protein degradation seen in permeabilized cells was representative of that seen in intact cells, as judged by the similar speed of degradation, substrate selectivity, temperature dependence, and involvement of free sulfhydryl groups. Degradation of these proteins in permeabilized cells took place in the absence of transport between the ER and the Golgi system. Moreover, degradation occurred in the absence of added ATP or cytosol, and in the presence of apyrase, GTP gamma S, or EDTA; i.e., under conditions which prevent transport of proteins out of the ER. The efficiency and selectivity of degradation of newly synthesized proteins were also conserved in an isolated ER fraction. These data indicate that the machinery responsible for pre-Golgi degradation of newly synthesized proteins exists within the ER itself, and can operate

  12. Blunting Autoantigen-induced FOXO3a Protein Phosphorylation and Degradation Is a Novel Pathway of Glucocorticoids for the Treatment of Systemic Lupus Erythematosus.

    PubMed

    Lu, Mudan; Xu, Wei; Gao, Bo; Xiong, Sidong

    2016-09-16

    Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting multiple organs. Glucocorticoids (GCs), the potent anti-inflammatory drugs, remain as a cornerstone in the treatment for SLE; nevertheless, their clinical efficacy is compromised by the side effects of long term treatment and resistance. To improve the therapeutic efficacy of GCs in SLE, it is important to further decipher the molecular mechanisms of how GCs exert their anti-inflammatory effects. In this investigation, FOXO3a was identified as a molecule that was down-regulated in the course of SLE. Of interest, GC treatment was found to rescue FOXO3a expression both in SLE mice and in SLE patients. Gain- and loss-of-function studies demonstrated that FOXO3a played a crucial role in GC treatment of SLE via inhibiting inflammatory responses. Further studies showed that the up-regulation of FOXO3a by GCs relied on the suppression of pI3K/AKT-mediated FOXO3a phosphorylation and the arrest of FOXO3a in the nucleus. Finally, our data revealed that FOXO3a was critical for GC-mediated inhibition of NF-κB activity, which might involve its interaction with NF-κB p65 protein. Collectively, these data indicated that FOXO3a played an important role in GC treatment of SLE by suppressing pro-inflammatory response, and targeting FOXO3a might provide a novel therapeutic strategy against SLE.

  13. Beef palatability and its relationship with protein degradation and muscle fibre type profile in longissimus thoracis in Alentejana breed from divergent growth pathways.

    PubMed

    Costa, P; Simões, J A; Alves, S P; Lemos, J P C; Alfaia, C M; Lopes, P A; Prates, J A M; Hocquette, J F; Calkins, C R; Vleck, V; Bessa, R J B

    2017-01-01

    The traditional beef production in the South of Portugal is based on a discontinuous growth (DG) system that requires lower external inputs and could enhance meat quality and financial returns to cattle producers. This system allows farmers to take advantage of the bull's compensatory growth when the pasture is abundant and finishes the cattle on concentrates for 2 to 3 months before slaughter. The fast gain rate before slaughter could be a valuable strategy to improve tenderness and to reduce its inconsistency in beef production. Therefore, the aim of this study was to evaluate the effects of production system (continuous growth (CG) v. DG) on longissimus thoracis muscle properties from Alentejana bulls. In total, 40 Alentejana male calves were allocated to two distinct feeding regimes: in the CG system, animals were fed concentrate plus hay and slaughtered at 18 months of age, whereas in the DG system, animals were fed on hay until 15 months of age and then fed the same diet provided to the CG group until 24 months of age. The DG system had a positive impact on meat tenderness (P<0.001) and global acceptability (P<0.001). DG bulls had greater fibre cross-sectional area (CSA) of glycolytic fibres (P<0.05) and relative area of the muscle (RA) occupied by type IIX fibres (P<0.01) and greater levels of α-actinin (P<0.05) and myosin light chain 2 (P<0.01) proteins, and pH24h (P<0.01) than CG bulls. The latter had greater CSA of type I (P<0.05) and type IIA (P<0.01) and greater RA of type IIA (P<0.05) and oxidative (P<0.05) than CG bulls. The compensatory growth production system had a positive impact on meat tenderness and global acceptability, overcoming the negative effects of slaughter of the bulls at a later age. The DG beef system could be a worthwhile strategy of beef production in Mediterranean areas due to the low-quality pasture in summer.

  14. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology.

    PubMed

    Guerriero, Christopher J; Brodsky, Jeffrey L

    2012-04-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding "problem," as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates.

  15. THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

    PubMed Central

    Guerriero, Christopher J.; Brodsky, Jeffrey L.

    2014-01-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

  16. Systematic Unraveling of the Unsolved Pathway of Nicotine Degradation in Pseudomonas

    PubMed Central

    Tang, Hongzhi; Zhang, Kunzhi; Yao, Yuxiang; Xu, Ping

    2013-01-01

    Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds. PMID:24204321

  17. Molecular characterization of the Akt-TOR signaling pathway in rainbow trout: potential role in muscle growth/degradation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Akt-TOR signaling pathway plays a key role in cellular metabolism and muscle growth. Hormone, nutrition and stress factors affect the Akt-TOR pathway by regulating gene transcription, protein synthesis and degradation. In addition, we previously showed that energetic demands elevate during vit...

  18. Degradation of Nicotine in Chlorinated Water: Pathways and ...

    EPA Pesticide Factsheets

    Report The objective of the study is to illustrate how drinking water would affect alkaloid pesticides, and to address the issue by (a) investigating the fate of nicotine in chlorinated drinking water and deionized water, (b) determining the reaction rate and pathway of the reaction between nicotine and aqueous chlorine, (c) identifying nicotine’s degradation products, and (d) providing data that can be used to assess the potential threat from nicotine in drinking water.

  19. Degradation of toluene-2,4-diamine by persulphate: kinetics, intermediates and degradation pathway.

    PubMed

    Jiang, Yong-hai; Zhang, Jin-bao; Xi, Bei-dou; An, Da; Yang, Yu; Li, Ming-xiao

    2015-01-01

    In this study, the degradation of toluene-2,4-diamine (TDA) by persulphate (PS) in an aqueous solution at near-neutral pH was examined. The result showed that the degradation rate of TDA increased with increasing PS concentrations. The optimal dosage of PS in the reaction system was determined by efficiency indicator (I) coupling in the consumption of PS and decay half-life of TDA. Calculation showed that 0.74 mM of PS was the most effective dosage for TDA degradation, at that level the maximum I of 24.51 was obtained. PS can oxidize TDA for an extended reaction time period. Under neutral condition without activation, four degradation intermediates, 2,4-diamino-3-hydroxy-5-sulfonicacidtoluene, 2,4-diaminobenzaldehyde, 2,4-bis(vinylamino)benzaldehyde and 3,5-diamino-4-hydroxy-2-pentene, were identified by high-performance liquid chromatography-mass spectrometry. The tentative degradation pathway of TDA was proposed as well. It was found that hydroxyl radical played an important role in degradation of TDA with the activation of Fe2+, whereas PS anion and sulphate radicals were responsible for the degradation without activation of Fe2+.

  20. Upregulation of macrophage-specific functions by oxidized LDL: lysosomal degradation-dependent and -independent pathways.

    PubMed

    Radhika, A; Sudhakaran, P R

    2013-01-01

    Formation of foam cells from macrophages, which are formed by the differentiation of blood-borne monocytes, is a critical early event in atherogenesis. To examine how pre-exposure of monocytes to modified proteins, such as oxLDL, influences their differentiation to macrophages, an in vitro model system using isolated PBMC maintained in culture in the presence of oxLDL was used. Pretreatment of monocytes with oxLDL caused a faster rate of expression of macrophage-specific functions and loss of monocyte-specific functions compared to unmodified LDL. The effect of oxidation of lipid component of LDL by CuSO(4) and its protein component by HOCl, on mo-mϕ differentiation was studied by monitoring the upregulation of macrophage-specific functions, particularly MMP-9. Chloroquine, a lysosomal degradation blocker, significantly reversed the effect mediated by CuSO(4) oxLDL, indicating the involvement of lysosomal degradation products, while no such effect was observed in HOCl oxLDL-treated cells, indicating the existence of a pathway independent of its lysosomal degradation products. Reversal of the effect of oxLDL by NAC and Calphostin C, an inhibitor of PKC, suggested the activation of RO-mediated signaling pathways. Use of inhibitors of signaling pathways showed that CuSO(4) oxLDL upregulated mϕ-specific MMP-9 through p38 MAPK and Akt-dependent pathways, while HOCl oxLDL utilized ERK ½ and Akt. Further analysis showed the activation of PPARγ and AP-1 in CuSO(4) oxLDL, while HOCl-oxLDL-mediated effect involved NFκB and AP-1. These results suggest that lipid oxLDL- and protein oxLDL-mediated upregulation of mo-mϕ-specific functions involve lysosomal degradation-dependent and -independent activation of intracellular signaling pathways.

  1. New hydrocarbon degradation pathways in the microbial metagenome from Brazilian petroleum reservoirs.

    PubMed

    Sierra-García, Isabel Natalia; Correa Alvarez, Javier; de Vasconcellos, Suzan Pantaroto; Pereira de Souza, Anete; dos Santos Neto, Eugenio Vaz; de Oliveira, Valéria Maia

    2014-01-01

    Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs.

  2. New Hydrocarbon Degradation Pathways in the Microbial Metagenome from Brazilian Petroleum Reservoirs

    PubMed Central

    Sierra-García, Isabel Natalia; Correa Alvarez, Javier; Pantaroto de Vasconcellos, Suzan; Pereira de Souza, Anete; dos Santos Neto, Eugenio Vaz; de Oliveira, Valéria Maia

    2014-01-01

    Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs. PMID:24587220

  3. Understanding protein glycosylation pathways in bacteria.

    PubMed

    Li, Hong; Debowski, Aleksandra W; Liao, Tingting; Tang, Hong; Nilsson, Hans-Olof; Marshall, Barry J; Stubbs, Keith A; Benghezal, Mohammed

    2017-01-01

    Through advances in analytical methods to detect glycoproteins and to determine glycan structures, there have been increasing reports of protein glycosylation in bacteria. In this review, we summarize the known pathways for bacterial protein glycosylation: lipid carrier-mediated 'en bloc' glycosylation; and cytoplasmic stepwise protein glycosylation. The exploitation of bacterial protein glycosylation systems, especially the 'mix and match' of three independent but similar pathways (oligosaccharyltransferase-mediated protein glycosylation, lipopolysaccharide and peptidoglycan biosynthesis) in Gram-negative bacteria for glycoengineering recombinant glycoproteins is also discussed.

  4. Elucidation of in situ polycyclic aromatic hydrocarbon degradation by functional metaproteomics (protein-SIP).

    PubMed

    Herbst, Florian-Alexander; Bahr, Arne; Duarte, Marcia; Pieper, Dietmar H; Richnow, Hans-Hermann; von Bergen, Martin; Seifert, Jana; Bombach, Petra

    2013-10-01

    Current knowledge of the physiology and phylogeny of polycyclic aromatic hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and isolations. In the present study, in situ microcosms consisting of activated carbon pellets (BACTRAP®s) were loaded with either (13) C-naphthalene or (13) C-fluorene and were subsequently exposed in the contaminant source and plume fringe region of a PAH-contaminated aquifer. Metaproteomic analysis and protein-stable isotope probing revealed Burkholderiales, Actinomycetales, and Rhizobiales as the most active microorganisms in the groundwater communities. Proteins identified of the naphthalene degradation pathway showed a relative (13) C isotope abundance of approximately 50 atom% demonstrating that the identified naphthalene-degrading bacteria gained at least 80% of their carbon by PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs showed a structure similar to the naphthalene-BACTRAPs, the identification of fluorene degraders and degradation pathways failed in situ. In complementary laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and possible fluorene degradation enzymes was observed. This result demonstrates the impact of laboratory conditions on microbial community structure and activity of certain species and underlines the need on in situ exploration of microbial community functions. In situ microcosms in combination with protein-stable isotope probing may be a significant tool for in situ identification of metabolic key players as well as degradation pathways.

  5. Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6-Dinitrotoluene

    PubMed Central

    Nishino, Shirley F.; Paoli, George C.; Spain, Jim C.

    2000-01-01

    An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol. PMID:10788393

  6. Degradation of ciprofloxacin in water by advanced oxidation process: kinetics study, influencing parameters and degradation pathways.

    PubMed

    Sayed, Murtaza; Ismail, M; Khan, Sanaullah; Tabassum, Safia; Khan, Hasan M

    2016-01-01

    Gamma-radiation-induced degradation of ciprofloxacin (CIP) in aqueous solution and the factors affecting the degradation process have been investigated. The results showed that CIP (4.6 mg/L) was almost completely degraded at an absorbed dose of 870 Gy. The kinetic studies of aqueous solutions containing 4.6, 10, 15 and 17.9 mg/L indicated that the decomposition of CIP by gamma irradiation followed pseudo-first-order kinetics and the decay constant (k) decreased from 5.9  ×  10(-3) to 1.6  ×  10(-3) Gy(-1) with an increase in CIP initial concentration from 4.6 to 17.9 mg/L. The effect of saturation of CIP solution with N2, N2O or air on radiation-induced degradation of CIP was also investigated. The effects of radical scavengers, such as t-BuOH and i-PrOH, showed the role of reactive radicals towards degradation of CIP in the order of OH > e(aq)- . H. The apparent second-order rate constant of [Formula: see text] with CIP was calculated to be 2.64 × 10(9) M(-1) s(-1). The effects of solution pH as well as natural water contaminants, such as [HCO3-, CO3(2-), and NO2-, on CIP degradation by gamma-irradiation were also investigated. Major degradation products, including organic acids, were identified using UPLC-MS/MS and IC, and degradation pathways have been proposed.

  7. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

    PubMed Central

    Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

    2016-01-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  8. The Degradation Pathway of the Mitophagy Receptor Atg32 Is Re-Routed by a Posttranslational Modification.

    PubMed

    Levchenko, Mariia; Lorenzi, Isotta; Dudek, Jan

    2016-01-01

    The outer mitochondrial membrane protein Atg32 is the central receptor for mitophagy, the mitochondria-specific form of autophagy. Atg32 is an unstable protein, and is rapidly degraded under conditions in which mitophagy is not induced. Here we show that Atg32 undergoes a posttranslational modification upon induction of mitophagy. The modification is dependent on the core autophagic machinery, including Atg8, and on the mitophagy-specific adaptor protein Atg11. The modified Atg32 is targeted to the vacuole where it becomes stabilized when vacuolar proteases are deficient. Interestingly, we find that this degradation pathway differs from the degradation pathway of non-modified Atg32, which neither involves vacuolar proteases, nor the proteasome. These analyses reveal that a posttranslational modification discriminates a form of Atg32 targeting mitochondria for mitophagy from that, which escapes mitophagy by rapid degradation.

  9. The Degradation Pathway of the Mitophagy Receptor Atg32 Is Re-Routed by a Posttranslational Modification

    PubMed Central

    Levchenko, Mariia; Lorenzi, Isotta

    2016-01-01

    The outer mitochondrial membrane protein Atg32 is the central receptor for mitophagy, the mitochondria-specific form of autophagy. Atg32 is an unstable protein, and is rapidly degraded under conditions in which mitophagy is not induced. Here we show that Atg32 undergoes a posttranslational modification upon induction of mitophagy. The modification is dependent on the core autophagic machinery, including Atg8, and on the mitophagy-specific adaptor protein Atg11. The modified Atg32 is targeted to the vacuole where it becomes stabilized when vacuolar proteases are deficient. Interestingly, we find that this degradation pathway differs from the degradation pathway of non-modified Atg32, which neither involves vacuolar proteases, nor the proteasome. These analyses reveal that a posttranslational modification discriminates a form of Atg32 targeting mitochondria for mitophagy from that, which escapes mitophagy by rapid degradation. PMID:27992522

  10. Genetic dissection of peroxisome-associated matrix protein degradation in Arabidopsis thaliana.

    PubMed

    Burkhart, Sarah E; Lingard, Matthew J; Bartel, Bonnie

    2013-01-01

    Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H(2)O(2), which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP-ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.

  11. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  12. Genomic and metabolic analysis of fluoranthene degradation pathway in Celeribacter indicus P73T

    PubMed Central

    Cao, Junwei; Lai, Qiliang; Yuan, Jun; Shao, Zongze

    2015-01-01

    Celeribacter indicus P73T, isolated from deep-sea sediment from the Indian Ocean, is capable of degrading a wide range of polycyclic aromatic hydrocarbons (PAHs) and is the first fluoranthene-degrading bacterium within the family Rhodobacteraceae. Here, the complete genome sequence of strain P73T is presented and analyzed. Besides a 4.5-Mb circular chromosome, strain P73T carries five plasmids, and encodes 4827 predicted protein-coding sequences. One hundred and thirty-eight genes, including 14 dioxygenase genes, were predicted to be involved in the degradation of aromatic compounds, and most of these genes are clustered in four regions. P73_0346 is the first fluoranthene 7,8-dioxygenase to be discovered and the first fluoranthene dioxygenase within the toluene/biphenyl family. The degradative genes in regions B and D in P73T are absent in Celeribacter baekdonensis B30, which cannot degrade PAHs. Four intermediate metabolites [acenaphthylene-1(2H)-one, acenaphthenequinone, 1,2-dihydroxyacenaphthylene, and 1,8-naphthalic anhydride] of fluoranthene degradation by strain P73T were detected as the main intermediates, indicating that the degradation of fluoranthene in P73T was initiated by dioxygenation at the C-7,8 positions. Based on the genomic and metabolitic results, we propose a C-7,8 dioxygenation pathway in which fluoranthene is mineralized to TCA cycle intermediates. PMID:25582347

  13. Interactions of Bacterial Proteins with Host Eukaryotic Ubiquitin Pathways

    PubMed Central

    Perrett, Charlotte Averil; Lin, David Yin-Wei; Zhou, Daoguo

    2011-01-01

    Ubiquitination is a post-translational modification in which one or more 76 amino acid polypeptide ubiquitin molecules are covalently linked to the lysine residues of target proteins. Ubiquitination is the main pathway for protein degradation that governs a variety of eukaryotic cellular processes, including the cell-cycle, vesicle trafficking, antigen presentation, and signal transduction. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many diseases including inflammatory and neurodegenerative disorders. Recent studies have revealed that viruses and bacterial pathogens exploit the host ubiquitination pathways to gain entry and to aid their survival/replication inside host cells. This review will summarize recent developments in understanding the biochemical and structural mechanisms utilized by bacterial pathogens to interact with the host ubiquitination pathways. PMID:21772834

  14. DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells.

    PubMed

    Zhang, Yinghao; Chang, Fang-Mei; Huang, Jianjun; Junco, Jacob J; Maffi, Shivani K; Pridgen, Hannah I; Catano, Gabriel; Dang, Hong; Ding, Xiang; Yang, Fuquan; Kim, Dae Joon; Slaga, Thomas J; He, Rongqiao; Wei, Sung-Jen

    2014-02-01

    Timely removal of oxidatively damaged proteins is critical for cells exposed to oxidative stresses; however, cellular mechanism for clearing oxidized proteins is not clear. Our study reveals a novel type of protein modification that may play a role in targeting oxidized proteins and remove them. In this process, DSS1 (deleted in split hand/split foot 1), an evolutionally conserved small protein, is conjugated to proteins induced by oxidative stresses in vitro and in vivo, implying oxidized proteins are DSS1 clients. A subsequent ubiquitination targeting DSS1-protein adducts has been observed, suggesting the client proteins are degraded through the ubiquitin-proteasome pathway. The DSS1 attachment to its clients is evidenced to be an enzymatic process modulated by an unidentified ATPase. We name this novel protein modification as DSSylation, in which DSS1 plays as a modifier, whose attachment may render target proteins a signature leading to their subsequent ubiquitination, thereby recruits proteasome to degrade them.

  15. Targeting the Autophagy/Lysosomal Degradation Pathway in Parkinson´s Disease

    PubMed Central

    Rivero-Ríos, Pilar; Madero-Pérez, Jesús; Fernández, Belén; Hilfiker, Sabine

    2016-01-01

    Autophagy is a cellular quality control mechanism crucial for neuronal homeostasis. Defects in autophagy are critically associated with mechanisms underlying Parkinson´s disease (PD), a common and debilitating neurodegenerative disorder. Autophagic dysfunction in PD can occur at several stages of the autophagy/lysosomal degradative machinery, contributing to the formation of intracellular protein aggregates and eventual neuronal cell death. Therefore, autophagy inducers may comprise a promising new therapeutic approach to combat neurodegeneration in PD. Several currently available FDA-approved drugs have been shown to enhance autophagy, which may allow for their repurposing for use in novel clinical conditions including PD. This review summarizes our current knowledge of deficits in the autophagy/lysosomal degradation pathways associated with PD, and highlight current approaches which target this pathway as possible means towards novel therapeutic strategies. PMID:26517050

  16. Aquatic photochemistry of isoflavone phytoestrogens: degradation kinetics and pathways.

    PubMed

    Felcyn, Jacob R; Davis, Jasmine C C; Tran, Loan H; Berude, John C; Latch, Douglas E

    2012-06-19

    Isoflavones are plant-derived chemicals that are potential endocrine disruptors. Although some recent studies have detected isoflavones in natural waters, little is known about their aquatic fates. The photochemical behaviors of the isoflavones daidzein, formononetin, biochanin A, genistein, and equol were studied under simulated solar light and natural sunlight. All of these phytoestrogens were found to be photolabile under certain conditions. Daidzein and formononetin degraded primarily by direct photolysis. Their expected near-surface summer half-lives in pH 7 water at 47° latitude are expected to be 10 and 4.6 h, respectively. Biochanin A, genistein, and equol degraded relatively slowly by direct photolysis at environmentally realistic pH values, though they showed significant degradation rate enhancements in the presence of natural organic matter (NOM). The indirect photolysis rates for these compounds scaled with NOM concentration, and NOM from microbial origin was found to be a more potent photosensitizer than NOM from terrestrial sources. Mechanistic studies were performed to determine the indirect photolysis pathways responsible for the rate enhancements. Results of these studies implicate reaction with both singlet oxygen and excited state triplet NOM. Environmental half-lives for biochanin A, genistein, and equol are expected to vary on the basis of pH as well as NOM source and concentration.

  17. Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

    PubMed Central

    Yun, Sung Ho; Choi, Chi-Won; Yi, Yoon-Sun; Kim, Jonghyun; Chung, Young-Ho; Park, Edmond Changkyun; Kim, Seung Il

    2016-01-01

    Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX. PMID:27124467

  18. Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24.

    PubMed

    Lee, Sang-Yeop; Kim, Gun-Hwa; Yun, Sung Ho; Choi, Chi-Won; Yi, Yoon-Sun; Kim, Jonghyun; Chung, Young-Ho; Park, Edmond Changkyun; Kim, Seung Il

    2016-01-01

    Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.

  19. Hydrolytic and oxidative degradation of electrospun supramolecular biomaterials: In vitro degradation pathways.

    PubMed

    Brugmans, M C P; Sӧntjens, S H M; Cox, M A J; Nandakumar, A; Bosman, A W; Mes, T; Janssen, H M; Bouten, C V C; Baaijens, F P T; Driessen-Mol, A

    2015-11-01

    The emerging field of in situ tissue engineering (TE) of load bearing tissues places high demands on the implanted scaffolds, as these scaffolds should provide mechanical stability immediately upon implantation. The new class of synthetic supramolecular biomaterial polymers, which contain non-covalent interactions between the polymer chains, thereby forming complex 3D structures by self assembly. Here, we have aimed to map the degradation characteristics of promising (supramolecular) materials, by using a combination of in vitro tests. The selected biomaterials were all polycaprolactones (PCLs), either conventional and unmodified PCL, or PCL with supramolecular hydrogen bonding moieties (either 2-ureido-[1H]-pyrimidin-4-one or bis-urea units) incorporated into the backbone. As these materials are elastomeric, they are suitable candidates for cardiovascular TE applications. Electrospun scaffold strips of these materials were incubated with solutions containing enzymes that catalyze hydrolysis, or solutions containing oxidative species. At several time points, chemical, morphological, and mechanical properties were investigated. It was demonstrated that conventional and supramolecular PCL-based polymers respond differently to enzyme-accelerated hydrolytic or oxidative degradation, depending on the morphological and chemical composition of the material. Conventional PCL is more prone to hydrolytic enzymatic degradation as compared to the investigated supramolecular materials, while, in contrast, the latter materials are more susceptible to oxidative degradation. Given the observed degradation pathways of the examined materials, we are able to tailor degradation characteristics by combining selected PCL backbones with additional supramolecular moieties. The presented combination of in vitro test methods can be employed to screen, limit, and select biomaterials for pre-clinical in vivo studies targeted to different clinical applications.

  20. Iodinated contrast media electro-degradation: process performance and degradation pathways.

    PubMed

    Del Moro, Guido; Pastore, Carlo; Di Iaconi, Claudio; Mascolo, Giuseppe

    2015-02-15

    The electrochemical degradation of six of the most widely used iodinated contrast media was investigated. Batch experiments were performed under constant current conditions using two DSA® electrodes (titanium coated with a proprietary and patented mixed metal oxide solution of precious metals such as iridium, ruthenium, platinum, rhodium and tantalum). The degradation removal never fell below 85% (at a current density of 64 mA/cm(2) with a reaction time of 150 min) when perchlorate was used as the supporting electrolyte; however, when sulphate was used, the degradation performance was above 80% (at a current density of 64 mA/cm(2) with a reaction time of 150 min) for all of the compounds studied. Three main degradation pathways were identified, namely, the reductive de-iodination of the aromatic ring, the reduction of alkyl aromatic amides to simple amides and the de-acylation of N-aromatic amides to produce aromatic amines. However, as amidotrizoate is an aromatic carboxylate, this is added via the decarboxylation reaction. The investigation did not reveal toxicity except for the lower current density used, which has shown a modest toxicity, most likely for some reaction intermediates that are not further degraded. In order to obtain total removal of the contrast media, it was necessary to employ a current intensity between 118 and 182 mA/cm(2) with energy consumption higher than 370 kWh/m(3). Overall, the electrochemical degradation was revealed to be a reliable process for the treatment of iodinated contrast media that can be found in contaminated waters such as hospital wastewater or pharmaceutical waste-contaminated streams.

  1. The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

    PubMed

    Zhao, Wenming; Guan, Chunmei; Feng, Jian; Liang, Yan; Zhan, Ni; Zuo, Jianru; Ren, Bo

    2016-07-01

    In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination.

  2. CBS9106-induced CRM1 degradation is mediated by cullin ring ligase activity and the neddylation pathway.

    PubMed

    Saito, Naoya; Sakakibara, Keiichi; Sato, Takuji; Friedman, Jonathan M; Kufe, Donald W; VonHoff, Daniel D; Kawabe, Takumi

    2014-12-01

    Chromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is overexpressed in various cancers. Recent studies have also reported that CRM1 protein expression is a negative prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1, decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However, the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3) or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation and G1 phase cell-cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1 degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition and apoptosis.

  3. Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells

    PubMed Central

    Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

    2016-01-01

    L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells. PMID:26983598

  4. Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation.

    PubMed

    Costaglioli, Patricia; Barthe, Christophe; Claverol, Stephane; Brözel, Volker S; Perrot, Michel; Crouzet, Marc; Bonneu, Marc; Garbay, Bertrand; Vilain, Sebastien

    2012-09-01

    Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.

  5. FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates its Delivery to the Vacuolar Degradation Pathway.

    PubMed

    Belda-Palazon, Borja; Rodriguez, Lesia; Fernandez, Maria A; Castillo, Mari-Cruz; Anderson, Erin A; Gao, Caiji; González-Guzmán, Miguel; Peirats-Llobet, Marta; Zhao, Qiong; De Winne, Nancy; Gevaert, Kris; De Jaeger, Geert; Jiang, Liwen; Leon, Jose; Mullen, Robert T; Rodriguez, Pedro L

    2016-08-05

    Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana. This suggested that ubiquitylated ABA receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knock-down fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA. Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.

  6. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  7. Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways

    SciTech Connect

    Haigler, B.E.; Spain, J.C. )

    1991-11-01

    Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. The authors have investigate the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of {sup 18}O{sub 2} indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1, 2-dihydroxy-3-nitrocyclohexa-3, 5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation.

  8. Balance Between Folding and Degradation for Hsp90-Dependent Client Proteins: A Key Role for CHIP

    PubMed Central

    Kundrat, Lenka; Regan, Lynne

    2011-01-01

    Cells must regulate the synthesis and degradation of their proteins to maintain a balance that is appropriate for their specific growth conditions. Here we present the results of an investigation of the balance between protein folding and degradation for mammalian chaperone Hsp90-dependent client proteins. The central players are the molecular chaperones Hsp70 and Hsp90, the co-chaperone HOP, and ubiquitin ligase, CHIP. Hsp70 and Hsp90 bind to HOP thus forming a ternary folding complex whereas the binding of CHIP to the chaperones has previously been shown to lead to ubiquitination and ultimately to degradation of the client proteins as well as the chaperones. To understand the folding/degradation balance in more detail, we characterized the stoichiometries of the CHIP-Hsp70 and CHIP-Hsp90 complexes and measured the corresponding dissociation constants to be ~ 1 µM and ~ 4.5 µM respectively. We quantified the rate of ubiquitination of various substrates by CHIP in vitro. We further determined that the folding and degradation machineries cannot coexist in one complex. Lastly, we measured the in vivo concentrations of Hsp70, Hsp90, HOP, and CHIP under normal conditions and when client proteins are being degraded due to inhibition of the folding pathway. These in vivo measurements along with the in vitro data allowed us to calculate the approximate cellular concentrations of the folding and degradation complexes under both conditions and formulate a quantitative model for the balance between protein folding and degradation as well as an explanation for the shift to client protein degradation when the folding pathway is inhibited. PMID:20704274

  9. Degradation of misfolded proteins by autophagy: is it a strategy for Huntington's disease treatment?

    PubMed

    Lin, Fang; Qin, Zheng-Hong

    2013-01-01

    Autophagy is a degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. The accumulation and aggregation of misfolded proteins are hallmarks of several neurodegenerative diseases. Many researchers have reported that autophagy degrades disease-causing misfolded and aggregated proteins, including mutant huntingtin (Htt) in Huntington's disease, mutant synuclein in familial Parkingson's disease, mutant Cu, Zn-Superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis. In this review, we will bring up new evidence to elucidate the involvement of autophagy in degradation of mutant Htt, discuss the mechanisms regulating the degradation of mutant Htt by autophagy and the therapeutic effects of drugs that enhance autophagy to improve clearance of mutant Htt. We propose that enhancement of autophagy by drugs may be a strategy to treat or retard progression of Huntington's disease.

  10. Roles of Protein Ubiquitination and Degradation Kinetics in Biological Oscillations

    PubMed Central

    Xu, Lida; Qu, Zhilin

    2012-01-01

    Protein ubiquitination and degradation play important roles in many biological functions and are associated with many human diseases. It is well known that for biochemical oscillations to occur, proper degradation rates of the participating proteins are needed. In most mathematical models of biochemical reactions, linear degradation kinetics has been used. However, the degradation kinetics in real systems may be nonlinear, and how nonlinear degradation kinetics affects biological oscillations are not well understood. In this study, we first develop a biochemical reaction model of protein ubiquitination and degradation and calculate the degradation rate against the concentration of the free substrate. We show that the protein degradation kinetics mainly follows the Michaelis-Menten formulation with a time delay caused by ubiquitination and deubiquitination. We then study analytically how the Michaelis-Menten degradation kinetics affects the instabilities that lead to oscillations using three generic oscillation models: 1) a positive feedback mediated oscillator; 2) a positive-plus-negative feedback mediated oscillator; and 3) a negative feedback mediated oscillator. In all three cases, nonlinear degradation kinetics promotes oscillations, especially for the negative feedback mediated oscillator, resulting in much larger oscillation amplitudes and slower frequencies than those observed with linear kinetics. However, the time delay due to protein ubiquitination and deubiquitination generally suppresses oscillations, reducing the amplitude and increasing the frequency of the oscillations. These theoretical analyses provide mechanistic insights into the effects of specific proteins in the ubiquitination-proteasome system on biological oscillations. PMID:22506034

  11. Ubiquitin in the peroxisomal protein import pathway.

    PubMed

    Francisco, Tânia; Rodrigues, Tony A; Pinto, Manuel P; Carvalho, Andreia F; Azevedo, Jorge E; Grou, Cláudia P

    2014-03-01

    PEX5 is the shuttling receptor for newly synthesized peroxisomal matrix proteins. Alone, or with the help of an adaptor protein, this receptor binds peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane docking/translocation module (DTM). The interaction between cargo-loaded PEX5 and the DTM ultimately results in its insertion into the DTM with the concomitant translocation of the cargo protein across the organelle membrane. PEX5 is not consumed in this event; rather it is dislocated back into the cytosol so that it can promote additional rounds of protein transportation. Remarkably, the data collected in recent years indicate that dislocation is preceded by monoubiquitination of PEX5 at a conserved cysteine residue. This mandatory modification is not the only type of ubiquitination occurring at the DTM. Indeed, several findings suggest that defective receptors jamming the DTM are polyubiquitinated and targeted to the proteasome for degradation.

  12. Protein export through the bacterial Sec pathway.

    PubMed

    Tsirigotaki, Alexandra; De Geyter, Jozefien; Šoštaric, Nikolina; Economou, Anastassios; Karamanou, Spyridoula

    2017-01-01

    The general secretory (Sec) pathway comprises an essential, ubiquitous and universal export machinery for most proteins that integrate into, or translocate through, the plasma membrane. Sec exportome polypeptides are synthesized as pre-proteins that have cleavable signal peptides fused to the exported mature domains. Recent advances have re-evaluated the interaction networks of pre-proteins with chaperones that are involved in pre-protein targeting from the ribosome to the SecYEG channel and have identified conformational signals as checkpoints for high-fidelity targeting and translocation. The recent structural and mechanistic insights into the channel and its ATPase motor SecA are important steps towards the elucidation of the allosteric crosstalk that mediates secretion. In this Review, we discuss recent biochemical, structural and mechanistic insights into the consecutive steps of the Sec pathway - sorting and targeting, translocation and release - in both co-translational and post-translational modes of export. The architecture and conformational dynamics of the SecYEG channel and its regulation by ribosomes, SecA and pre-proteins are highlighted. Moreover, we present conceptual models of the mechanisms and energetics of the Sec-pathway dependent secretion process in bacteria.

  13. Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway.

    PubMed

    Lallemand-Breitenbach, Valérie; Jeanne, Marion; Benhenda, Shirine; Nasr, Rihab; Lei, Ming; Peres, Laurent; Zhou, Jun; Zhu, Jun; Raught, Brian; de Thé, Hugues

    2008-05-01

    In acute promyelocytic leukaemia (APL), arsenic trioxide induces degradation of the fusion protein encoded by the PML-RARA oncogene, differentiation of leukaemic cells and produces clinical remissions. SUMOylation of its PML moiety was previously implicated, but the nature of the degradation pathway involved and the role of PML-RARalpha catabolism in the response to therapy have both remained elusive. Here, we demonstrate that arsenic-induced PML SUMOylation triggers its Lys 48-linked polyubiquitination and proteasome-dependent degradation. When exposed to arsenic, SUMOylated PML recruits RNF4, the human orthologue of the yeast SUMO-dependent E3 ubiquitin-ligase, as well as ubiquitin and proteasomes onto PML nuclear bodies. Arsenic-induced differentiation is impaired in cells transformed by a non-degradable PML-RARalpha SUMOylation mutant or in APL cells transduced with a dominant-negative RNF4, directly implicating PML-RARalpha catabolism in the therapeutic response. We thus identify PML as the first protein degraded by SUMO-dependent polyubiquitination. As PML SUMOylation recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, these domains could physically integrate the SUMOylation, ubiquitination and degradation pathways.

  14. Fenton degradation of Cartap hydrochloride: identification of the main intermediates and the degradation pathway.

    PubMed

    Tian, Kaixun; Ming, Cuixiang; Dai, Youzhi; Honore Ake, Kouassi Marius

    2015-01-01

    The advanced oxidation of Cartap hydrochloride (Cartap) promoted by the Fenton system in an aqueous medium was investigated. Based on total organic carbon, chemical oxygen demand and high-performance liquid chromatography, the oxidation of Cartap is quite efficient by the Fenton system. Its long chain is easily destroyed, but the reaction does not proceed to complete mineralization. Ion chromatography detection indicated the formation of acetic acid, propionic acid, formic acid, nitrous acid and sulfuric acid in the reaction mixtures. Further evidence of nitrogen monoxide and sulfur dioxide formation was obtained by using a flue gas analyzer. Monitoring by gas chromatograph-mass spectrometer demonstrated the formation of oxalic acid, ethanol, carbon dioxide, and L-alanine ethylamide. Based on these experimental results, plausible degradation pathways for Cartap mineralization in an aqueous medium by the Fenton system are proposed.

  15. ISG12a Restricts Hepatitis C Virus Infection through the Ubiquitination-Dependent Degradation Pathway

    PubMed Central

    Xue, Binbin; Yang, Darong; Wang, Jingjing; Xu, Yan; Wang, Xiaohong; Qin, Yuwen; Tian, Renyun; Chen, Shengwen; Xie, Qinya; Liu, Nianli

    2016-01-01

    ABSTRACT Interferons (IFNs) restrict various kinds of viral infection via induction of hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high-IFN-inducible gene, ISG12a (also known as IFI27), exhibits a nonapoptotic antiviral effect on hepatitis C virus (HCV) infection. Viral NS5A protein is targeted specifically by ISG12a, which mediates NS5A degradation via a ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. S-phase kinase-associated protein 2 (SKP2) is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity. IMPORTANCE Upon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadly unclear. The present study shows a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase, SKP2, for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity. PMID:27194766

  16. The nature of protein folding pathways

    PubMed Central

    Englander, S. Walter; Mayne, Leland

    2014-01-01

    How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The protein folding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure. PMID:25326421

  17. A New 4-Nitrotoluene Degradation Pathway in a Mycobacterium Strain

    PubMed Central

    Spiess, Tilmann; Desiere, Frank; Fischer, Peter; Spain, Jim C.; Knackmuss, Hans-Joachim; Lenke, Hiltrud

    1998-01-01

    Mycobacterium sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the Mycobacterium strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-m-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-m-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-m-cresol, observed during growth of the Mycobacterium strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (λmax, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-m-cresol by meta ring cleavage. PMID:9464378

  18. Pathway Analysis Incorporating Protein-Protein Interaction Networks Identified Candidate Pathways for the Seven Common Diseases

    PubMed Central

    Lin, Peng-Lin; Yu, Ya-Wen

    2016-01-01

    Pathway analysis has become popular as a secondary analysis strategy for genome-wide association studies (GWAS). Most of the current pathway analysis methods aggregate signals from the main effects of single nucleotide polymorphisms (SNPs) in genes within a pathway without considering the effects of gene-gene interactions. However, gene-gene interactions can also have critical effects on complex diseases. Protein-protein interaction (PPI) networks have been used to define gene pairs for the gene-gene interaction tests. Incorporating the PPI information to define gene pairs for interaction tests within pathways can increase the power for pathway-based association tests. We propose a pathway association test, which aggregates the interaction signals in PPI networks within a pathway, for GWAS with case-control samples. Gene size is properly considered in the test so that genes do not contribute more to the test statistic simply due to their size. Simulation studies were performed to verify that the method is a valid test and can have more power than other pathway association tests in the presence of gene-gene interactions within a pathway under different scenarios. We applied the test to the Wellcome Trust Case Control Consortium GWAS datasets for seven common diseases. The most significant pathway is the chaperones modulate interferon signaling pathway for Crohn’s disease (p-value = 0.0003). The pathway modulates interferon gamma, which induces the JAK/STAT pathway that is involved in Crohn’s disease. Several other pathways that have functional implications for the seven diseases were also identified. The proposed test based on gene-gene interaction signals in PPI networks can be used as a complementary tool to the current existing pathway analysis methods focusing on main effects of genes. An efficient software implementing the method is freely available at http://puppi.sourceforge.net. PMID:27622767

  19. Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

    SciTech Connect

    Chou, Bin; Hiromatsu, Kenji; Hisaeda, Hajime; Duan, Xuefeng; Imai, Takashi; Murata, Shigeo; Tanaka, Keiji; Himeno, Kunisuke

    2010-02-12

    Cytotoxic CD8{sup +} T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8{sup +} T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8{sup +} T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4{sup +} T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8{sup +} T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.

  20. Regulation of the p73 protein stability and degradation

    SciTech Connect

    Oberst, Andrew; Salomoni, Paolo; Pandolfi, Pier Paolo; Oren, Moshe; Melino, Gerry; Bernassola, Francesca . E-mail: bernasso@uniroma2.it

    2005-06-10

    p73, a homologue to the tumor suppressor gene p53, is involved in tumorigenesis, though its specific role remains unclear. The gene has two distinct promoters which allow the formation of two protein isoforms with opposite effects: full-length transactivating (TA) p73 shows pro-apoptotic effects, while the shorter {delta}Np73, which lacks the N-terminal transactivating domain, has an evident anti-apoptotic function. Unlike p53, the p73 gene is rarely mutated in human cancers. However, alterations in the relative levels of TA and {delta}Np73 have been shown to correlate with prognosis in several human cancers, suggesting that the fine regulation of these two isoforms is of pivotal importance in controlling proliferation and cell death. Much effort is currently focused on the elucidation of the mechanisms that differentially control TA and {delta}Np73 activity and protein stability, a process complicated by the finding that both proteins are regulated by a similar suite of complex post-translational modifications that include ubiquitination, sequential phosphorylation, prolyl-isomerization, recruitment into the PML-nuclear body (PML-NB), and acetylation. Here we shall consider the main regulatory partners of p73, with particular attention to the recently discovered Itch- and Nedd8-mediated degradation pathways, along with the emerging roles of PML, p38 MAP kinase, Pin1, and p300 in p73 transcriptional activation, and possible mechanisms for the differential regulation of the TAp73 and {delta}Np73 isoforms.

  1. The non-phagocytic route of collagen uptake: a distinct degradation pathway.

    PubMed

    Madsen, Daniel H; Ingvarsen, Signe; Jürgensen, Henrik J; Melander, Maria C; Kjøller, Lars; Moyer, Amanda; Honoré, Christian; Madsen, Charlotte A; Garred, Peter; Burgdorf, Sven; Bugge, Thomas H; Behrendt, Niels; Engelholm, Lars H

    2011-07-29

    The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.

  2. Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development.

    PubMed

    Li, Lei; Nelson, Clark J; Trösch, Josua; Castleden, Ian; Huang, Shaobai; Millar, A Harvey

    2017-02-01

    We applied (15)N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with (15)N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome.

  3. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    PubMed

    Carrión, Cristian A; Martínez, Dana E; Costa, M Lorenza; Guiamet, Juan José

    2014-11-11

    Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, "senescence-associated vacuoles" (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves.

  4. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    PubMed Central

    Carrión, Cristian A.; Martínez, Dana E.; Costa, M. Lorenza; Guiamet, Juan José

    2014-01-01

    Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, “senescence-associated vacuoles” (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves. PMID:27135516

  5. Hepatitis B Virus Protein X Induces Degradation of Talin-1

    PubMed Central

    van de Klundert, Maarten A. A.; van den Biggelaar, Maartje; Kootstra, Neeltje A.; Zaaijer, Hans L.

    2016-01-01

    In the infected human hepatocyte, expression of the hepatitis B virus (HBV) accessory protein X (HBx) is essential to maintain viral replication in vivo. HBx critically interacts with the host damaged DNA binding protein 1 (DDB1) and the associated ubiquitin ligase machinery, suggesting that HBx functions by inducing the degradation of host proteins. To identify such host proteins, we systematically analyzed the HBx interactome. One HBx interacting protein, talin-1 (TLN1), was proteasomally degraded upon HBx expression. Further analysis showed that TLN1 levels indeed modulate HBV transcriptional activity in an HBx-dependent manner. This indicates that HBx-mediated TLN1 degradation is essential and sufficient to stimulate HBV replication. Our data show that TLN1 can act as a viral restriction factor that suppresses HBV replication, and suggest that the HBx relieves this restriction by inducing TLN1 degradation. PMID:27775586

  6. Presenilin-2 regulates the degradation of RBP-Jk protein through p38 mitogen-activated protein kinase.

    PubMed

    Kim, Su-Man; Kim, Mi-Yeon; Ann, Eun-Jung; Mo, Jung-Soon; Yoon, Ji-Hye; Park, Hee-Sae

    2012-03-01

    Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk)--a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.

  7. RFP tags for labeling secretory pathway proteins

    SciTech Connect

    Han, Liyang; Zhao, Yanhua; Xu, Pingyong; Huan, Shuangyan

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

  8. Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids

    PubMed Central

    1983-01-01

    Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids. PMID:6853596

  9. Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

    PubMed

    Skop, V; Cahová, M; Papáčková, Z; Páleníčková, E; Daňková, H; Baranowski, M; Zabielski, P; Zdychová, J; Zídková, J; Kazdová, L

    2012-01-01

    We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

  10. Optimum folding pathways for growing protein chains.

    PubMed

    Senturk, Serife; Baday, Sefer; Arkun, Yaman; Erman, Burak

    2007-11-26

    The folding of a protein is studied as it grows residue by residue from the N-terminus and enters an environment that stabilizes the folded state. This mode of folding of a growing chain is different from refolding where the full chain folds from a disordered initial configuration to the native state. We propose a sequential dynamic optimization method that computes the evolution of optimum folding pathways as amino acid residues are added to the peptide chain one by one. The dynamic optimization formulation is deterministic and uses Newton's equations of motion and a Go-type potential that establishes the native contacts and excluded volume effects. The method predicts the optimal energy-minimizing path among all the alternative feasible pathways. As two examples, the folding of the chicken villin headpiece, a 36-residue protein, and chymotrypsin inhibitor 2 (CI2), a 64-residue protein, are studied. Results on the villin headpiece show significant differences from the refolding of the same chain studied previously. Results on CI2 mostly agree with the results of refolding experiments and computational work.

  11. Occurrence, leaching, and degradation of Cry1Ab protein from transgenic maize detritus in agricultural streams

    DOE PAGES

    Griffiths, Natalie A.; Tank, Jennifer L.; Royer, Todd V.; ...

    2017-03-15

    The insecticidal Cry1Ab protein expressed by transgenic (Bt) maize can enter adjacent water bodies via multiple pathways, but its fate in stream ecosystems is not as well studied as in terrestrial systems. In this study, we used a combination of field sampling and laboratory experiments to examine the occurrence, leaching, and degradation of soluble Cry1Ab protein derived from Bt maize in agricultural streams. We surveyed 11 agricultural streams in northwestern Indiana, USA, on 6 dates that encompassed the growing season, crop harvest, and snowmelt/spring flooding, and detected Cry1Ab protein in the water column and in flowing subsurface tile drains atmore » concentrations of 3–60 ng/L. In a series of laboratory experiments, submerged Bt maize leaves leached Cry1Ab into stream water with 1% of the protein remaining in leaves after 70 d. Laboratory experiments suggested that dissolved Cry1Ab protein degraded rapidly in microcosms containing water-column microorganisms, and light did not enhance breakdown by stimulating assimilatory uptake of the protein by autotrophs. Here, the common detection of Cry1Ab protein in streams sampled across an agricultural landscape, combined with laboratory studies showing rapid leaching and degradation, suggests that Cry1Ab may be pseudo-persistent at the watershed scale due to the multiple input pathways from the surrounding terrestrial environment.« less

  12. Hydroxide Degradation Pathways for Substituted Benzyltrimethyl Ammonium: A DFT Study

    DOE PAGES

    Long, Hai; Pivovar, Bryan S.

    2014-11-01

    The stability of cations used in the alkaline exchange membranes has been a major challenge. In this paper, degradation energy barriers were investigated by density functional theory for substituted benzyltrimethyl ammonium (BTMA+) cations. Findings show that electron-donating substituent groups at meta-position(s) of the benzyl ring could result in increased degradation barriers. However, after investigating more than thirty substituted BTMA+ cations, the largest improvement in degradation barrier found was only 6.7 kJ/mol. This suggests a modest (8×) improvement in stability for this type of approach may be possible, but for anything greater other approaches will need to be pursued.

  13. Degradation of microinjected proteins: the role of substrate flexibility

    SciTech Connect

    Rote, K.V.

    1985-01-01

    RB-mediated microinjection was used to introduce radioiodinated proteins of similar structure, but diverse flexibilities, into HeLa cells. Rates of intracellular degradation were then measured by release of /sup 125/I-tyrosine into the media. Ribonuclease-A was much more stable to degradation by trypsin, pepsin, or papain than its relatively flexible derivatives ribonuclease-S and S-protein. Likewise, ribonuclease-S and S-protein were degraded more quickly in reticulocyte lysates than ribonuclease-A. In contrast, all three proteins displayed similar, if not identical, half-lives in vivo. Similarly, intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were in the same range whether they were measured in the free state or following complex formation, which drastically decreases flexibility. Trypsinogen, which contains a relatively flexible activation domain, was degraded more slowly than anhydrotrypsin. Nondenaturing agarose or polyacrylamide gel electrophoresis of microinjected cell lysates revealed that complexes of trypsin and its inhibitors remained intact following radioiodination and introduction into cells, and are therefore degraded as a unit. All microinjected proteins remained in their unbound, unprocessed forms prior to degradation.

  14. The Homogentisate and Homoprotocatechuate Central Pathways Are Involved in 3- and 4-Hydroxyphenylacetate Degradation by Burkholderia xenovorans LB400

    PubMed Central

    Méndez, Valentina; Agulló, Loreine; González, Myriam; Seeger, Michael

    2011-01-01

    Background Genome characterization of the model PCB-degrading bacterium Burkholderia xenovorans LB400 revealed the presence of eleven central pathways for aromatic compounds degradation, among them, the homogentisate and the homoprotocatechuate pathways. However, the functionality of these central pathways in strain LB400 has not been assessed and related peripheral pathways has not been described. Methodology/Principal Findings The aims of this study were to determine the functionality of the homogentisate and homoprotocatechuate central pathways in B. xenovorans LB400 and to establish their role in 3-hydroxyphenylacetate (3-HPA) and 4-hydroxyphenylacetate (4-HPA) catabolism. Strain LB400 was able to grow using 3-HPA and 4-HPA as sole carbon source. A genomic search in LB400 suggested the presence of mhaAB and hpaBC genes clusters encoding proteins of the 3-hydroxyphenylacetate and 4-hydroxyphenylacetate peripheral pathways. LB400 cells grown with 3-HPA and 4-HPA degraded homogentisate and homoprotocatechuate and showed homogentisate 1,2-dioxygenase and homoprotocatechuate 2,3-dioxygenase activities. Transcriptional analyses by RT-PCR showed the expression of two chromosomally-encoded homogentisate dioxygenases (BxeA2725 and BxeA3900) and the hpaD gene encoding the homoprotocatechuate 2,3-dioxygenase during 3-HPA and 4-HPA degradation. The proteome analyses by two-dimensional polyacrilamide gel electrophoresis of B. xenovorans LB400 grown in 3-HPA and 4-HPA showed the induction of fumarylacetoacetate hydrolase HmgB (BxeA3899). Conclusions/Significance This study revealed that strain LB400 used both homogentisate and homoprotocatechuate ring-cleavage pathways for 3- hydroxyphenylacetate and 4-hydroxyphenylacetate catabolism and that these four catabolic routes are functional, confirming the metabolic versatility of B. xenovorans LB400. PMID:21423751

  15. Identification of Genes and Pathways Related to Phenol Degradation in Metagenomic Libraries from Petroleum Refinery Wastewater

    PubMed Central

    Silva, Cynthia C.; Hayden, Helen; Sawbridge, Tim; Mele, Pauline; De Paula, Sérgio O.; Silva, Lívia C. F.; Vidigal, Pedro M. P.; Vicentini, Renato; Sousa, Maíra P.; Torres, Ana Paula R.; Santiago, Vânia M. J.; Oliveira, Valéria M.

    2013-01-01

    Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system. PMID:23637911

  16. The protein C pathway and sepsis.

    PubMed

    Della Valle, Patrizia; Pavani, Giulia; D'Angelo, Armando

    2012-03-01

    After the discovery of the key components of the protein C (PC) pathway a beneficial effect on survival of the infusion of activated protein C (APC) in animal models of sepsis was demonstrated, leading to the development of recombinant human activated protein C (rh-APC) as a therapeutic agent. It soon became clear that rather than the anticoagulant and profibrinolytic activities of APC, its anti-inflammatory and cytoprotective properties played a major role in the treatment of patients with severe sepsis. Such properties affect the response to inflammation of endothelial cells and leukocytes and are exerted through binding of APC to at least five receptors with intracellular signaling. The main APC protective mechanism involves binding of the Gla-domain to the endothelial protein C receptor (EPCR) and cleavage of protease activated receptor 1 (PAR-1), eliciting suppression of proinflammatory cytokines synthesis and of intracellular proapoptotic pathways and activation of endothelial barrier properties. However, thrombin cleaves PAR-1 with much higher catalytic efficiency, followed by pro-inflammatory, pro-apoptotic and barrier disruptive intracellular signaling, and it is unclear how APC can exert a protective activity through the cleavage of PAR-1 when thrombin is also present in the same environment. Interestingly, in endothelial cell cultures, PAR-1 cleavage by thrombin results in anti-inflammatory and barrier protective signaling provided occupation of EPCR by the PC gla-domain, raising the possibility that the beneficial effects of rh-APC might be recapitulated in vivo by administration of h-PC zymogen to patients with severe sepsis. Recent reports of h-PC infusion in animal models of sepsis support this hypothesis.

  17. Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

    PubMed Central

    Fang, Nancy N.; Zhu, Mang; Rose, Amalia; Wu, Kuen-Phon; Mayor, Thibault

    2016-01-01

    Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions. PMID:27698423

  18. Proteasomal degradation of beta-carotene metabolite--modified proteins.

    PubMed

    Sommerburg, Olaf; Karius, Nicole; Siems, Werner; Langhans, Claus-Dieter; Leichsenring, Michael; Breusing, Nicolle; Grune, Tilman

    2009-01-01

    Free radical attack on beta-carotene results in the formation of high amounts of carotene breakdown products (CBPs) having biological activities. As several of the CBPs are reactive aldehydes, it has to be considered that these compounds are able to modify proteins. Therefore, the aim of the study was to investigate whether CBP-modification of proteins is leading to damaged proteins recognized and degraded by the proteasomal system. We used the model proteins tau and ferritin to test whether CBPs will modify them and whether such modifications lead to enhanced proteasomal degradation. To modify proteins, we used crude CBPs as a mixture obtained after hypochloric acid derived BC degradation, as well as several single compounds, as apo8'-carotenal, retinal, or beta-ionone. The majority of the CBPs found in our reaction mixture are well known metabolites as described earlier after BC degradation using different oxidants. CBPs are able to modify proteins, and in in vitro studies, we were able to demonstrate that the 20S proteasome is able to recognize and degrade CBP-modified proteins preferentially. In testing the proteolytic response of HT22 cells toward CBPs, we could demonstrate an enhanced protein turnover, which is sensitive to lactacystin. Interestingly, the proteasomal activity is resistant to treatment with CBP. On the other hand, we were able to demonstrate that supraphysiological levels of CBPs might lead to the formation of protein-CBP-adducts that are able to inhibit the proteasome. Therefore, the removal of CBP-modified proteins seems to be catalyzed by the proteasomal system and is effective, if the formation of CBPs is not overwhelming and leading to protein aggregates.

  19. Cycle inhibiting factors (cifs): cyclomodulins that usurp the ubiquitin-dependent degradation pathway of host cells.

    PubMed

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Oswald, Eric

    2011-04-01

    Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are "cyclomodulins" that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

  20. Suppression of Cartilage Degradation by Zingerone Involving the p38 and JNK MAPK Signaling Pathway.

    PubMed

    Ruangsuriya, Jetsada; Budprom, Piyaporn; Viriyakhasem, Nawarat; Kongdang, Patiwat; Chokchaitaweesuk, Chatchadawalai; Sirikaew, Nutnicha; Chomdej, Siriwadee; Nganvongpanit, Korakot; Ongchai, Siriwan

    2017-02-01

    Zingerone, an active compound that is present in cooked ginger, has been claimed to be a bioactive ingredient that holds the potential of preventing and/or treating diseases involving inflammation. In this study, zingerone was used to discover its properties against joint inflammation using interleukin-1β-induced osteoarthritis in cartilage explant and cell culture models. Zingerone was supplemented into the cartilage explant and cell culture media at different concentrations along with the presence of interleukin-1β, an inducer of osteoarthritis. Markers indicating cartilage degradation, inflammation, and the signaling molecules involved in the inflammatory induction were investigated. Diacerien, an anti-osteoarthritic drug, was used as a positive control. Zingerone at a concentration of 40 µM reduced the level of matrix metalloproteinase-13 to about 31.95 ± 4.33 % compared with the interleukin-1β-treated group and halted cartilage explant degradation as indicated by reducing the accumulative release of sulfated glycosaminoglycans by falling to the control concomitantly with an elevation of the remaining contents of uronic acid and collagen in the explant tissues when zingerone was added. In the SW1353 cell line model, zingerone efficiently suppressed the expression of TNF-α, interleukin-6, and interleukin-8 mRNA levels and tended to reduce the levels of both p38 and c-Jun N-terminal kinase phosphorylation. From the results of this study, it can be concluded that zingerone potentially reduced cartilage degradation, which is partially involved in p38 and c-Jun N-terminal kinases of the mitogen activator protein kinase signaling pathway leading to the reduction of proinflammatory cytokine amplification effects and cartilage-degrading enzyme syntheses. This finding supports the contention that ginger holds positive pharmaceutical effects against osteoarthritis.

  1. In silico prediction of pharmaceutical degradation pathways: a benchmarking study.

    PubMed

    Kleinman, Mark H; Baertschi, Steven W; Alsante, Karen M; Reid, Darren L; Mowery, Mark D; Shimanovich, Roman; Foti, Chris; Smith, William K; Reynolds, Dan W; Nefliu, Marcela; Ott, Martin A

    2014-11-03

    Zeneth is a new software application capable of predicting degradation products derived from small molecule active pharmaceutical ingredients. This study was aimed at understanding the current status of Zeneth's predictive capabilities and assessing gaps in predictivity. Using data from 27 small molecule drug substances from five pharmaceutical companies, the evolution of Zeneth predictions through knowledge base development since 2009 was evaluated. The experimentally observed degradation products from forced degradation, accelerated, and long-term stability studies were compared to Zeneth predictions. Steady progress in predictive performance was observed as the knowledge bases grew and were refined. Over the course of the development covered within this evaluation, the ability of Zeneth to predict experimentally observed degradants increased from 31% to 54%. In particular, gaps in predictivity were noted in the areas of epimerizations, N-dealkylation of N-alkylheteroaromatic compounds, photochemical decarboxylations, and electrocyclic reactions. The results of this study show that knowledge base development efforts have increased the ability of Zeneth to predict relevant degradation products and aid pharmaceutical research. This study has also provided valuable information to help guide further improvements to Zeneth and its knowledge base.

  2. Xkid Is Degraded in a D-Box, KEN-Box, and A-Box-Independent Pathway

    PubMed Central

    Castro, Anna; Vigneron, Suzanne; Bernis, Cyril; Labbé, Jean-Claude; Lorca, Thierry

    2003-01-01

    During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC. PMID:12773557

  3. Xkid is degraded in a D-box, KEN-box, and A-box-independent pathway.

    PubMed

    Castro, Anna; Vigneron, Suzanne; Bernis, Cyril; Labbé, Jean-Claude; Lorca, Thierry

    2003-06-01

    During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC.

  4. Degradation of diclofenac by UV-activated persulfate process: Kinetic studies, degradation pathways and toxicity assessments.

    PubMed

    Lu, Xian; Shao, Yisheng; Gao, Naiyun; Chen, Juxiang; Zhang, Yansen; Xiang, Huiming; Guo, Youluo

    2017-03-21

    Diclofenac (DCF) is the frequently detected non-steroidal pharmaceuticals in the aquatic environment. In this study, the degradation of DCF was evaluated by UV-254nm activated persulfate (UV/PS). The degradation of DCF followed the pseudo first-order kinetics pattern. The degradation rate constant (kobs) was accelerated by UV/PS compared to UV alone and PS alone. Increasing the initial PS dosage or solution pH significantly enhanced the degradation efficiency. Presence of various natural water constituents had different effects on DCF degradation, with an enhancement or inhibition in the presence of inorganic anions (HCO3(-) or Cl(-)) and a significant inhibition in the presence of NOM. In addition, preliminary degradation mechanisms and major products were elucidated using LC-MS/MS. Hydroxylation, decarbonylation, ring-opening and cyclation reaction involving the attack of SO4(•)(-) or other substances, were the main degradation mechanism. TOC analyzer and Microtox bioassay were employed to evaluate the mineralization and cytotoxicity of solutions treated by UV/PS at different times, respectively. Limited elimination of TOC (32%) was observed during the mineralization of DCF. More toxic degradation products and their related intermediate species were formed, and the UV/PS process was suitable for removing the toxicity. Of note, longer degradation time may be considered for the final toxicity removal.

  5. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination

    PubMed Central

    Braten, Ori; Livneh, Ido; Ziv, Tamar; Admon, Arie; Kehat, Izhak; Caspi, Lilac H.; Gonen, Hedva; Bercovich, Beatrice; Godzik, Adam; Jahandideh, Samad; Jaroszewski, Lukasz; Sommer, Thomas; Kwon, Yong Tae; Guharoy, Mainak; Tompa, Peter; Ciechanover, Aaron

    2016-01-01

    The “canonical” proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established—in both human and yeast cells—a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes. PMID:27385826

  6. Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination.

    PubMed

    Takasugi, Toshiyuki; Minegishi, Seiji; Asada, Akiko; Saito, Taro; Kawahara, Hiroyuki; Hisanaga, Shin-ichi

    2016-02-26

    Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death.

  7. Proteolysis-Targeting Chimeras: Induced Protein Degradation as a Therapeutic Strategy.

    PubMed

    Ottis, Philipp; Crews, Craig M

    2017-03-20

    Until recently, the only ways to reduce specific protein signaling were to either knock down the target by RNAi or to interfere with the signaling by inhibiting an enzyme or receptor within the signal transduction cascade. Herein, we review an emerging class of small molecule pharmacological agents, called PROTACs, that present a novel approach to specifically target proteins and their respective signaling pathways. These heterobifunctional molecules utilize endogenous cellular quality control machinery by recruiting it to target proteins in order to induce their degradation.

  8. ORGANOPHOSPHORUS PESTICIDE DEGRADATION PATHWAYS DURING DRINKING WATER TREATMENT

    EPA Science Inventory

    The objective of this work was to investigate organophosphorus (OP) pesticide transformation pathways as a class in the presence of aqueous chlorine. Seven priority OP pesticides were examined for their reactivity with aqueous chlorine: chlorpyrifos (CP), parathion (PA), diazino...

  9. Regulation of protein degradation in muscle by calcium

    NASA Technical Reports Server (NTRS)

    Zeman, Richard J.; Kameyama, Tsuneo; Matsumoto, Kazue; Bernstein, Paul; Etlinger, Joseph D.

    1985-01-01

    Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. The effect of different classes of protease inhibitors was tested to determine the responsible proteolytic systems involved in calcium-dependent degradation. The results suggest that nonlysosomal leupetin- and E-64-c-sensitive proteases are resposible for calcium-dependent proteolysis in muscle.

  10. Prion protein degradation by lichens of the genus Cladonia

    USGS Publications Warehouse

    Bennett, James P.; Rodriguez, Cynthia M.; Johnson, Christopher J.

    2012-01-01

    It has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species of Cladonia lichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity in Cladonia species did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.

  11. Mechanism and pathways of chlorfenapyr photocatalytic degradation in aqueous suspension of TiO2.

    PubMed

    Cao, Yongsong; Yi, Lei; Huang, Lu; Hou, Ying; Lu, Yitong

    2006-05-15

    The light-induced degradation of chlorfenapyr under UV was investigated in aqueous solutions containing TiO2 as photocatalyst. The photocatalytic degradation of chlorfenapyr followed pseudo-first-order degradation kinetics (Ct = C0e(-kt)). The study focused on the identification of possible intermediate products during the degradation, using gas chromatography mass-spectrometry (GC-MS) and 1HNMR. Six aromatic intermediates were identified by several techniques during the treatment and some of them were further confirmed by matching authentic standards. Structure analysis of the degradation products suggested two degradation pathways: (1) The aliphatic ether group was cleaved from chlorfenapyr to form pyrrole-alph-carboxylic acid, then the pyrrole group was broken to form 4-chloroglycine; (2) Chlorfenapyr was debrominated and the aliphatic ether group was cleaved from the pyrrole group, which was further broken to form 4-chlorophenylglycine. The glycine was degraded into 4-chlorobenzoic acids, which was further broken into inorganic ions and CO2.

  12. Bioenergetics and pathway of acid blue 113 degradation by Staphylococcus lentus.

    PubMed

    Sekar, Sudharshan; Mahadevan, Surianarayanan; Shanmugam, Bhuvanesh Kumar; Mandal, Asit Baran

    2012-01-01

    Bioreaction calorimetric studies of degradation of the dye acid blue 113 by Staphylococcus lentus are reported for the first time. The heat released during the dye degradation process can be successfully measured using reaction calorimeter. Power time and oxygen uptake rate (OUR) profile followed each other suggesting that heat profiles could monitor the progress of the dye degradation in biocalorimetry. The shifts observed in power-time profile indicated three distinct phases of the bioprocess indicating simultaneous utilization of glucose (primary) and dye (secondary carbon source). Secretion of azoreductase enzyme enhanced the degradation process. Optimization of aeration and agitation rates was observed to be vital to efficient dye degradation. The degradative pathway for acid blue 113 by S. lentus was delineated via high-performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FT-IR), and gas chromatography coupled with mass spectrometry (GC-MS) analyses. Interestingly the products of degradation were found to have low toxicity, as per cytotoxicity measurements.

  13. Synergetic effect of alkaline earth metal oxides and iron oxides on the degradation of hexachlorobenzene and its degradation pathway.

    PubMed

    Su, Guijin; Liu, Yexuan; Huang, Linyan; Shi, Yali; Zhang, Aiqian; Zhang, Lixia; Liu, Wenbin; Gao, Lirong; Zheng, Minghui

    2013-01-01

    The degradation of hexachlorobenzene (HCB) was carried out over physical mixtures of a series of alkaline earth metal oxides (MO: M=Mg, Ca, Sr, Ba) and iron oxides with different crystal types (Fe(x)O(y):Fe(2)O(3) or Fe(3)O(4)) at 300°C. These physical mixtures all showed a synergetic effect toward the degradation of HCB. A range of degradation products were identified by various methods, including tri- to penta-chlorobenzenes by gas chromatography/mass spectrometry (GC-MS), tri- to penta-chlorophenols, tetrachlorocatechol (TCC) and tetrachlorohydroquinone (TCHQ) by GC-MS after derivatization, and formic and acetic acids by ion chromatography. Two degradation pathways, hydrodechlorination and oxidative degradation, appear to occur competitively. However, more sequential chlorinated benzene and phenol congeners were formed over mixed MO/Fe(3)O(4) than over mixed MO/Fe(2)O(3) under the same conditions. The oxidative reaction dominated over mixed MO/Fe(2)O(3) and was promoted as the major reaction by the synergetic effect, while both the oxidative and hydrodechlorination reactions were important over mixed MO/Fe(3)O(4), and both pathways are remarkably promoted by the synergetic effect. The enhanced hydrodechlorination may be attributed to free electrons generated by the transformation of Fe(3)O(4) into Fe(2)O(3), and hydrogen provided by water adsorbed on the MO.

  14. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    PubMed

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

    2015-06-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  15. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Cancer.gov

    The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

  16. Small-Molecule PROTACS: New Approaches to Protein Degradation.

    PubMed

    Toure, Momar; Crews, Craig M

    2016-02-05

    The current inhibitor-based approach to therapeutics has inherent limitations owing to its occupancy-based model: 1) there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition, 2) high in vivo concentrations bring potential for off-target side effects, and 3) there is a need to bind to an active site, thus limiting the drug target space. As an alternative, induced protein degradation lacks these limitations. Based on an event-driven model, this approach offers a novel catalytic mechanism to irreversibly inhibit protein function by targeting protein destruction through recruitment to the cellular quality control machinery. Prior protein degrading strategies have lacked therapeutic potential. However, recent reports of small-molecule-based proteolysis-targeting chimeras (PROTACs) have demonstrated that this technology can effectively decrease the cellular levels of several protein classes.

  17. d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

    PubMed Central

    Johnsen, Ulrike; Dambeck, Michael; Zaiss, Henning; Fuhrer, Tobias; Soppa, Jörg; Sauer, Uwe; Schönheit, Peter

    2009-01-01

    The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following 13C-labeling patterns of proteinogenic amino acids after growth on [13C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus. PMID:19584053

  18. Aβ mediates Sigma receptor degradation via CaN/NFAT pathway

    PubMed Central

    Fang, Min; Zhang, Pei; Zhao, Yanxin; Jin, Aiping; Liu, Xueyuan

    2016-01-01

    Sigma receptor is an endoplasmic reticulum protein and belongs to non-opioid receptor. Increasing evidence shows that Sigma receptor activation can significantly attenuate AD induced neurological dysfunction and the functional deficiency of Sigma receptor plays an important role in the Aβ induced neuronal loss. This study aimed to investigate the influence of extracellular accumulation of Aβ on the Sigma receptor expression. Our results showed the increase in extracellular Aβ had little influence on the mRNA expression of Sigma receptor, but gradually reduced its protein expression. Co-immunoprecipitation was employed to evaluate the interaction of Sigma receptor with other proteins. Results showed BIP could bind to Sigma receptor to affect the ubiquitination of Sigma receptor. Further investigation showed there was a NFAT binding site at the promoter of BIP. Then, Western blot assay was performed to detect NFAT expression. Results showed extracellular Aβ affected the nuclear translocation of NFAT and the CaN activity of NFAT also increased with the accumulation of extracellular Aβ. In this study, NFAT-BIP luciferase reporter gene system was constructed. Results showed NFAT was able to regulate the transcription of BIP. Thus, we speculate that extracellular Aβ accumulation may activate CaN/NFAT signaling pathway to induce chaperone BIP expression, which results in Sigma receptor ubiquitination and its degradation. PMID:27648137

  19. M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

    PubMed Central

    Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

    2013-01-01

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

  20. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway.

    PubMed

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S; Brenner, David A; Burgdorf, Sven; Engelholm, Lars H; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto; Bugge, Thomas H

    2013-09-16

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process.

  1. Development of target protein-selective degradation inducer for protein knockdown.

    PubMed

    Itoh, Yukihiro; Ishikawa, Minoru; Kitaguchi, Risa; Sato, Shinichi; Naito, Mikihiko; Hashimoto, Yuichi

    2011-05-15

    Our previous technique for inducing selective degradation of target proteins with ester-type SNIPER (Specific and Nongenetic Inhibitor-of-apoptosis-proteins (IAPs)-dependent Protein ERaser) degrades both the target proteins and IAPs. Here, we designed a small-molecular amide-type SNIPER to overcome this issue. As proof of concept, we synthesized and biologically evaluated an amide-type SNIPER which induces selective degradation of cellular retinoic acid binding protein II (CRABP-II), but not IAPs. Such small-molecular, amide-type SNIPERs that induce target protein-selective degradation without affecting IAPs should be effective tools to study the biological roles of target proteins in living cells.

  2. PathwayMatrix: visualizing binary relationships between proteins in biological pathways

    PubMed Central

    2015-01-01

    Background Molecular activation pathways are inherently complex, and understanding relations across many biochemical reactions and reaction types is difficult. Visualizing and analyzing a pathway is a challenge due to the network size and the diversity of relations between proteins and molecules. Results In this paper, we introduce PathwayMatrix, a visualization tool that presents the binary relations between proteins in the pathway via the use of an interactive adjacency matrix. We provide filtering, lensing, clustering, and brushing and linking capabilities in order to present relevant details about proteins within a pathway. Conclusions We evaluated PathwayMatrix by conducting a series of in-depth interviews with domain experts who provided positive feedback, leading us to believe that our visualization technique could be helpful for the larger community of researchers utilizing pathway visualizations. PathwayMatrix is freely available at https://github.com/CreativeCodingLab/PathwayMatrix. PMID:26361499

  3. Control of alternative splicing by signal-dependent degradation of splicing-regulatory proteins.

    PubMed

    Katzenberger, Rebeccah J; Marengo, Matthew S; Wassarman, David A

    2009-04-17

    Alternative pre-mRNA splicing is a major gene expression regulatory mechanism in metazoan organisms. Proteins that bind pre-mRNA elements and control assembly of splicing complexes regulate utilization of pre-mRNA alternative splice sites. To understand how signaling pathways impact this mechanism, an RNA interference screen in Drosophila S2 cells was used to identify proteins that regulate TAF1 (TBP-associated factor 1) alternative splicing in response to activation of the ATR (ATM-RAD3-related) signaling pathway by the chemotherapeutic drug camptothecin (CPT). The screen identified 15 proteins that, when knocked down, caused the same change in TAF1 alternative splicing as CPT treatment. However, combined RNA interference and CPT treatment experiments indicated that only a subset of the identified proteins are targets of the CPT-induced signal, suggesting that multiple independent pathways regulate TAF1 alternative splicing. To understand how signals modulate the function of splicing factors, we characterized one of the CPT targets, Tra2 (Transformer-2). CPT was found to down-regulate Tra2 protein levels. CPT-induced Tra2 down-regulation was ATR-dependent and temporally paralleled the change in TAF1 alternative splicing, supporting the conclusion that Tra2 directly regulates TAF1 alternative splicing. Additionally, CPT-induced Tra2 down-regulation occurred independently of new protein synthesis, suggesting a post-translational mechanism. The proteasome inhibitor MG132 reduced CPT-induced Tra2 degradation and TAF1 alternative splicing, and mutation of evolutionarily conserved Tra2 lysine 81, a potential ubiquitin conjugation site, to arginine inhibited CPT-induced Tra2 degradation, supporting a proteasome-dependent alternative splicing mechanism. We conclude that CPT-induced TAF1 alternative splicing occurs through ATR-signaled degradation of a subset of splicing-regulatory proteins.

  4. PHOSPHOLIPIDS OF FIVE PSEUDOMONAD ARCHETYPES FOR DIFFERENT TOLUENE DEGRADATION PATHWAYS

    EPA Science Inventory

    Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was used to determine phospholipid profiles for five reference pseudomonad strains harboring distinct toluene catabolic pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, B...

  5. Pathways for degradation of plastic polymers floating in the marine environment.

    PubMed

    Gewert, Berit; Plassmann, Merle M; MacLeod, Matthew

    2015-09-01

    Each year vast amounts of plastic are produced worldwide. When released to the environment, plastics accumulate, and plastic debris in the world's oceans is of particular environmental concern. More than 60% of all floating debris in the oceans is plastic and amounts are increasing each year. Plastic polymers in the marine environment are exposed to sunlight, oxidants and physical stress, and over time they weather and degrade. The degradation processes and products must be understood to detect and evaluate potential environmental hazards. Some attention has been drawn to additives and persistent organic pollutants that sorb to the plastic surface, but so far the chemicals generated by degradation of the plastic polymers themselves have not been well studied from an environmental perspective. In this paper we review available information about the degradation pathways and chemicals that are formed by degradation of the six plastic types that are most widely used in Europe. We extrapolate that information to likely pathways and possible degradation products under environmental conditions found on the oceans' surface. The potential degradation pathways and products depend on the polymer type. UV-radiation and oxygen are the most important factors that initiate degradation of polymers with a carbon-carbon backbone, leading to chain scission. Smaller polymer fragments formed by chain scission are more susceptible to biodegradation and therefore abiotic degradation is expected to precede biodegradation. When heteroatoms are present in the main chain of a polymer, degradation proceeds by photo-oxidation, hydrolysis, and biodegradation. Degradation of plastic polymers can lead to low molecular weight polymer fragments, like monomers and oligomers, and formation of new end groups, especially carboxylic acids.

  6. The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP

    PubMed Central

    Lin, Ni-Hsuan; Huang, Yu-Shan; Opal, Puneet; Goldman, Robert D.; Messing, Albee; Perng, Ming-Der

    2016-01-01

    Alexander disease (AxD) is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding the intermediate filament (IF) protein GFAP. This disease is characterized by excessive accumulation of GFAP, known as Rosenthal fibers, within astrocytes. Abnormal GFAP aggregation also occurs in giant axon neuropathy (GAN), which is caused by recessive mutations in the gene encoding gigaxonin. Given that one of the functions of gigaxonin is to facilitate proteasomal degradation of several IF proteins, we sought to determine whether gigaxonin is involved in the degradation of GFAP. Using a lentiviral transduction system, we demonstrated that gigaxonin levels influence the degradation of GFAP in primary astrocytes and in cell lines that express this IF protein. Gigaxonin was similarly involved in the degradation of some but not all AxD-associated GFAP mutants. In addition, gigaxonin directly bound to GFAP, and inhibition of proteasome reversed the clearance of GFAP in cells achieved by overexpressing gigaxonin. These studies identify gigaxonin as an important factor that targets GFAP for degradation through the proteasome pathway. Our findings provide a critical foundation for future studies aimed at reducing or reversing pathological accumulation of GFAP as a potential therapeutic strategy for AxD and related diseases. PMID:27798231

  7. Eukaryotic starch degradation: integration of plastidial and cytosolic pathways.

    PubMed

    Fettke, Joerg; Hejazi, Mahdi; Smirnova, Julia; Höchel, Erik; Stage, Marion; Steup, Martin

    2009-01-01

    Starch is an important plant product widely used as a nutrient, as a source of renewable energy, and for many technological applications. In plants, starch is the almost ubiquitous storage carbohydrate whereas most heterotrophic prokaryotes and eukaryotes rely on glycogen. Despite close similarities in basic chemical features, starch and glycogen differ in both structural and physicochemical properties. Glycogen is a hydrosoluble macromolecule with evenly distributed branching points. Starch exists as a water-insoluble particle having a defined (and evolutionary conserved) internal structure. The biochemistry of starch requires the co-operation of up to 40 distinct (iso)enzymes whilst approximately 10 (iso)enzymes permit glycogen metabolism. The biosynthesis and degradation of native starch include the transition of carbohydrates from the soluble to the solid phase and vice versa. In this review, two novel aspects of the eukaryotic plastidial starch degradation are discussed: Firstly, biochemical reactions that take place at the surface of particulate glucans and mediate the phase transition of carbohydrates. Secondly, processes that occur downstream of the export of starch-derived sugars into the cytosol. Degradation of transitory starch mainly results in the formation of neutral sugars, such as glucose and maltose, that are transported into the cytosol via the respective translocators. The cytosolic metabolism of the neutral sugars includes the action of a hexokinase, a phosphoglucomutase, and a transglucosidase that utilizes high molecular weight glycans as a transient glucosyl acceptor or donor. Data are included on the transglucosidase (disproportionating isozyme 2) in Cyanophora paradoxa that accumulates storage carbohydrates in the cytosol rather than in the plastid.

  8. Regulation of protein degradation by insulin-degrading enzyme: analysis by small interfering RNA-mediated gene silencing.

    PubMed

    Fawcett, Janet; Permana, Paska A; Levy, Jennifer L; Duckworth, William C

    2007-12-01

    Proteins are vital to the overall structure of cells and to the function of cells in the form of enzymes. Thus the control of protein metabolism is among the most important aspects of cellular metabolism. Insulin's major effect on protein metabolism in the adult animal is inhibition of protein degradation. This is via inhibition of proteasome activity via an interaction with insulin-degrading enzyme (IDE). IDE is responsible for the majority of cellular insulin degradation. We hypothesized that a reduction in IDE would reduce insulin degradation and insulin's ability to inhibit protein degradation. HepG2 cells were transfected with siRNA against human IDE and insulin degradation and protein degradation measured. Both IDE mRNA and protein were reduced by >50% in the IDE siRNA transfected cells. Insulin degradation was reduced by approximately 50%. Cells were labeled with [3H]-leucine to investigate protein degradation. Short-lived protein degradation was unchanged in the cells with reduced IDE expression. Long-lived and very-long-lived protein degradation was reduced in the cells with reduced IDE expression (14.0+/-0.16 vs. 12.5+/-0.07%/4h (long-lived), 9.6+/-2.2% vs. 7.3+/-0.2%/3h (very-long-lived), control vs. IDE transfected, respectively, P<0.005). The inhibition of protein degradation by insulin was reduced 37-76% by a decreased expression of IDE in HepG2 cells. This shows that IDE is involved in cellular insulin metabolism and provides further evidence that insulin inhibits protein degradation via an interaction with IDE.

  9. Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation.

    PubMed

    Amaya, Celina; Militello, Rodrigo D; Calligaris, Sebastián D; Colombo, María I

    2016-11-01

    Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome-lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co-localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA-Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ-BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein-sorting (HOPS) complex hampered the co-localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway.

  10. Pathways and substrate-specific regulation of amino acid degradation in Phaeobacter inhibens DSM 17395 (archetype of the marine Roseobacter clade).

    PubMed

    Drüppel, Katharina; Hensler, Michael; Trautwein, Kathleen; Koßmehl, Sebastian; Wöhlbrand, Lars; Schmidt-Hohagen, Kerstin; Ulbrich, Marcus; Bergen, Nils; Meier-Kolthoff, Jan P; Göker, Markus; Klenk, Hans-Peter; Schomburg, Dietmar; Rabus, Ralf

    2014-01-01

    Combining omics and enzymatic approaches, catabolic routes of nine selected amino acids (tryptophan, phenylalanine, methionine, leucine, isoleucine, valine, histidine, lysine and threonine) were elucidated in substrate-adapted cells of Phaeobacter inhibens DSM 17395 (displaying conspicuous morphotypes). The catabolic network [excluding tricarboxylic acid (TCA) cycle] was reconstructed from 71 genes (scattered across the chromosome; one-third newly assigned), with 69 encoded proteins and 20 specific metabolites identified, and activities of 10 different enzymes determined. For example, Ph. inhibens DSM 17395 does not degrade lysine via the widespread saccharopine pathway but might rather employ two parallel pathways via 5-aminopentanoate or 2-aminoadipate. Tryptophan degradation proceeds via kynurenine and 2-aminobenzoate; the latter is metabolized as known from Azoarcus evansii. Histidine degradation is analogous to the Pseudomonas-type Hut pathway via N-formyl-l-glutamate. For threonine, only one of the three genome-predicted degradation pathways (employing threonine 3-dehydrogenase) is used. Proteins of the individual peripheral degradation sequences in Ph. inhibens DSM 17395 were apparently substrate-specifically formed contrasting the non-modulated TCA cycle enzymes. Comparison of genes for the reconstructed amino acid degradation network in Ph. inhibens DSM 17395 across 27 other complete genomes of Roseobacter clade members revealed most of them to be widespread among roseobacters.

  11. Induced protein degradation: an emerging drug discovery paradigm.

    PubMed

    Lai, Ashton C; Crews, Craig M

    2017-02-01

    Small-molecule drug discovery has traditionally focused on occupancy of a binding site that directly affects protein function, and this approach typically precludes targeting proteins that lack such amenable sites. Furthermore, high systemic drug exposures may be needed to maintain sufficient target inhibition in vivo, increasing the risk of undesirable off-target effects. Induced protein degradation is an alternative approach that is event-driven: upon drug binding, the target protein is tagged for elimination. Emerging technologies based on proteolysis-targeting chimaeras (PROTACs) that exploit cellular quality control machinery to selectively degrade target proteins are attracting considerable attention in the pharmaceutical industry owing to the advantages they could offer over traditional small-molecule strategies. These advantages include the potential to reduce systemic drug exposure, the ability to counteract increased target protein expression that often accompanies inhibition of protein function and the potential ability to target proteins that are not currently therapeutically tractable, such as transcription factors, scaffolding and regulatory proteins.

  12. Photocatalytic degradation of methylene blue with a nanocomposite system: synthesis, photocatalysis and degradation pathways.

    PubMed

    Xia, Shengjie; Zhang, Lianyang; Pan, Guoxiang; Qian, Pingping; Ni, Zheming

    2015-02-21

    Three different composites, including a calcined FeOOH supported ZnAl layered double hydroxide (FeOOH-LDO), a calcined ZnAl layered double hydroxide (ZnAl-LDO) and a calcined ZnFeAl layered double hydroxide (ZnFeAl-LDO), were synthesized via a sol-gel method, and their activity for the visible light photocatalytic degradation of methylene blue (MB) was studied. The composites were characterized by PXRD, SEM, and BET techniques, confirming the formation of highly crystalline structures. The activity performance of MB degradation was in the following order: FeOOH-LDO (∼95%) > ZnFeAl-LDO (∼60%) > ZnAl-LDO (∼23%). In addition, a possible photocatalytic degradation reaction mechanism for MB was also proposed. Moreover, the frontier electron densities on the atoms of MB were calculated, which were in satisfactory agreement with the postulated mechanism.

  13. Degradation of sulfonamide antibiotics by Microbacterium sp. strain BR1 - elucidating the downstream pathway.

    PubMed

    Ricken, Benjamin; Fellmann, Oliver; Kohler, Hans-Peter E; Schäffer, Andreas; Corvini, Philippe François-Xavier; Kolvenbach, Boris Alexander

    2015-12-25

    Microbacterium sp. strain BR1 is among the first bacterial isolates which were proven to degrade sulfonamide antibiotics. The degradation is initiated by an ipso-substitution, initiating the decay of the molecule into sulfur dioxide, the substrate specific heterocyclic moiety as a stable metabolite and benzoquinone imine. The latter appears to be instantaneously reduced to p-aminophenol, as that in turn was detected as the first stable intermediate. This study investigated the downstream pathway of sulfonamide antibiotics by testing the strain's ability to degrade suspected intermediates of this pathway. While p-aminophenol was degraded, degradation products could not be identified. Benzoquinone was shown to be degraded to hydroquinone and hydroquinone in turn was shown to be degraded to 1,2,4-trihydroxybenzene. The latter is assumed to be the potential substrate for aromatic ring cleavage. However, no products from the degradation of 1,2,4-trihydroxybenzene could be identified. There are no signs of accumulation of intermediates causing oxidative stress, which makes Microbacterium sp. strain BR1 an interesting candidate for industrial waste water treatment.

  14. Phenol degradation by Sulfobacillus acidophilus TPY via the meta-pathway.

    PubMed

    Zhou, Wengen; Guo, Wenbin; Zhou, Hongbo; Chen, Xinhua

    2016-09-01

    Due to its toxicity and volatility, phenol must be cleared from the environment. Sulfobacillus acidophilus TPY, which was isolated from a hydrothermal vent in the Pacific Ocean as a moderately thermoacidophilic Gram-positive bacterium, was capable of aerobically degrading phenol. This bacterium could tolerate up to 1300mg/L phenol and degrade 100mg/L phenol in 40h completely at 45°C and pH 1.8 with a maximal degradation rate of 2.32mg/L/h at 38h. Genome-wide search revealed that one gene (TPY_3176) and 14 genes clustered together in two regions with locus tags of TPY_0628-0634 and TPY_0640-0646 was proposed to be involved in phenol degradation via the meta-pathway with both the 4-oxalocrotonate branch and the hydrolytic branch. Real-time PCR analysis of S. acidophilus TPY under phenol cultivation condition confirmed the transcription of proposed genes involved in the phenol degradation meta-pathway. Degradation of 3-methylphenol and 2-methylphenol confirmed that the hydrolytic branch was utilised by S. acidophilus TPY. Phylogenetic analysis revealed that S. acidophilus TPY was closely related to sulphate-reducing bacteria and some Gram-positive phenol-degrading bacteria. This was the first report demonstrating the ability of S. acidophilus to degrade phenol and characterising the putative genes involved in phenol metabolism in S. acidophilus TPY.

  15. REGγ regulates ERα degradation via ubiquitin–proteasome pathway in breast cancer

    SciTech Connect

    Chai, Fan; Liang, Yan; Bi, Jiong; Chen, Li; Zhang, Fan; Cui, Youhong; Jiang, Jun

    2015-01-02

    Highlights: • High expression of REGγ is correlated with ERα status and poor clinical features. • Cell growth, mobility and invasion are significantly impaired by REGγ knockdown. • REGγ indirectly regulates ERα protein expression. - Abstract: REGγ is a proteasome coactivator which regulates proteolytic activity in eukaryotic cells. Abundant lines of evidence have showed that REGγ is over expressed in a number of human carcinomas. However, its precise role in the pathogenesis of cancer is still unclear. In this study, by examining 200 human breast cancer specimens, we demonstrated that REGγ was highly expressed in breast cancers, and the expression of REGγ was positively correlated with breast cancer patient estrogen receptor alpha (ERα) status. Moreover, the expression of REGγ was found positively associated with poor clinical features and low survival rates in ERα positive breast cancer patients. Further cell culture studies using MCF7 and BT474 breast cancer cell lines showed that cell proliferation, motility, and invasion capacities were decreased significantly by REGγ knockdown. Lastly, we demonstrated that REGγ indirectly regulates the degradation of ERα protein via ubiquitin–proteasome pathway. In conclusion, our findings provide the evidence that REGγ expression was positively correlated with ERα status and poor clinical prognosis in ERα positive breast cancer patients. As well, we disclose a new connection between the two molecules that are both highly expressed in most breast cancer cases.

  16. The F-box protein FBXO25 promotes the proteasome-dependent degradation of ELK-1 protein.

    PubMed

    Teixeira, Felipe R; Manfiolli, Adriana O; Soares, Cláudia S; Baqui, Munira M A; Koide, Tie; Gomes, Marcelo D

    2013-09-27

    FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.

  17. Unveiling New Degradation Intermediates/Pathways from the Photocatalytic Degradation of Microcystin-LR

    EPA Science Inventory

    This study focuses on the identification of reaction intermediates formed during the photocatalytic degradation of the cyanotoxin microcystin-LR with immobilized TiO2 Tphotocatalysts at neutral pH. To differentiate between impurities already existing in the MC-LR stand...

  18. The senescence-induced staygreen protein regulates chlorophyll degradation.

    PubMed

    Park, So-Yon; Yu, Jae-Woong; Park, Jong-Sung; Li, Jinjie; Yoo, Soo-Cheul; Lee, Na-Yeoun; Lee, Sang-Kyu; Jeong, Seok-Won; Seo, Hak Soo; Koh, Hee-Jong; Jeon, Jong-Seong; Park, Youn-Il; Paek, Nam-Chon

    2007-05-01

    Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the staygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase-impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf stay-greenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana benthamiana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively.

  19. Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

    PubMed

    Eshraghi, Aria; Dixon, Shandee D; Tamilselvam, Batcha; Kim, Emily Jin-Kyung; Gargi, Amandeep; Kulik, Julia C; Damoiseaux, Robert; Blanke, Steven R; Bradley, Kenneth A

    2014-07-01

    Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

  20. Temperature compensation via cooperative stability in protein degradation

    NASA Astrophysics Data System (ADS)

    Peng, Yuanyuan; Hasegawa, Yoshihiko; Noman, Nasimul; Iba, Hitoshi

    2015-08-01

    Temperature compensation is a notable property of circadian oscillators that indicates the insensitivity of the oscillator system's period to temperature changes; the underlying mechanism, however, is still unclear. We investigated the influence of protein dimerization and cooperative stability in protein degradation on the temperature compensation ability of two oscillators. Here, cooperative stability means that high-order oligomers are more stable than their monomeric counterparts. The period of an oscillator is affected by the parameters of the dynamic system, which in turn are influenced by temperature. We adopted the Repressilator and the Atkinson oscillator to analyze the temperature sensitivity of their periods. Phase sensitivity analysis was employed to evaluate the period variations of different models induced by perturbations to the parameters. Furthermore, we used experimental data provided by other studies to determine the reasonable range of parameter temperature sensitivity. We then applied the linear programming method to the oscillatory systems to analyze the effects of protein dimerization and cooperative stability on the temperature sensitivity of their periods, which reflects the ability of temperature compensation in circadian rhythms. Our study explains the temperature compensation mechanism for circadian clocks. Compared with the no-dimer mathematical model and linear model for protein degradation, our theoretical results show that the nonlinear protein degradation caused by cooperative stability is more beneficial for realizing temperature compensation of the circadian clock.

  1. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  2. Loss of protein association causes cardiolipin degradation in Barth syndrome

    PubMed Central

    Xu, Yang; Phoon, Colin K.L.; Berno, Bob; D’Souza, Kenneth; Hoedt, Esthelle; Zhang, Guoan; Neubert, Thomas A.; Epand, Richard M.; Ren, Mindong; Schlame, Michael

    2016-01-01

    Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but in Barth syndrome, where this association is compromised, cardiolipin becomes unstable, which causes the accumulation of monolyso-cardiolipin. PMID:27348092

  3. Structural basis of PROTAC cooperative recognition for selective protein degradation.

    PubMed

    Gadd, Morgan S; Testa, Andrea; Lucas, Xavier; Chan, Kwok-Ho; Chen, Wenzhang; Lamont, Douglas J; Zengerle, Michael; Ciulli, Alessio

    2017-03-13

    Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4(BD2)). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4(BD2). Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.

  4. BC-box protein domain-related mechanism for VHL protein degradation

    PubMed Central

    Pozzebon, Maria Elena; Varadaraj, Archana; Mattoscio, Domenico; Jaffray, Ellis G.; Miccolo, Claudia; Galimberti, Viviana; Tommasino, Massimo; Hay, Ronald T.; Chiocca, Susanna

    2013-01-01

    The tumor suppressor VHL (von Hippel–Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins. PMID:24145437

  5. Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

    SciTech Connect

    Pohlschroder Mechthild

    2009-02-03

    Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tat pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.

  6. Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells.

    PubMed

    Yao, Kang; Yin, Yulong; Li, Xilong; Xi, Pengbin; Wang, Junjun; Lei, Jian; Hou, Yongqing; Wu, Guoyao

    2012-06-01

    α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.

  7. Has the Bacterial Biphenyl Catabolic Pathway Evolved Primarily To Degrade Biphenyl? The Diphenylmethane Case

    PubMed Central

    Pham, Thi Thanh My

    2013-01-01

    In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl analogs in which the phenyl rings are bonded to a single carbon. Both chemicals are of environmental concern. P. pnomenusa B356 grew well on diphenylmethane. On the basis of growth kinetics analyses, diphenylmethane and biphenyl were shown to induce the same catabolic pathway. The profile of metabolites produced during growth of strain B356 on diphenylmethane was the same as the one produced by isolated enzymes of the biphenyl catabolic pathway acting individually or in coupled reactions. The biphenyl dioxygenase oxidizes diphenylmethane to 3-benzylcyclohexa-3,5-diene-1,2-diol very efficiently, and ultimately this metabolite is transformed to phenylacetic acid, which is further metabolized by a lower pathway. Strain B356 was also able to cometabolize benzophenone through its biphenyl pathway, although in this case, this substrate was unable to induce the biphenyl catabolic pathway and the degradation was incomplete, with accumulation of 2-hydroxy-6,7-dioxo-7-phenylheptanoic acid. Unlike strain B356, B. xenovorans LB400 did not grow on diphenylmethane. Its biphenyl pathway enzymes metabolized diphenylmethane, but they poorly metabolize benzophenone. The fact that the biphenyl catabolic pathway of strain B356 metabolized diphenylmethane and benzophenone more efficiently than that of strain LB400 brings us to postulate that in strain B356, this pathway evolved divergently to serve other functions not related to biphenyl degradation. PMID:23749969

  8. Stuxnet Facilitates the Degradation of Polycomb Protein during Development.

    PubMed

    Du, Juan; Zhang, Junzheng; He, Tao; Li, Yajuan; Su, Ying; Tie, Feng; Liu, Min; Harte, Peter J; Zhu, Alan Jian

    2016-06-20

    Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene, stuxnet (stx), that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Our biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity.

  9. Terrestrial and marine perspectives on modeling organic matter degradation pathways.

    PubMed

    Burd, Adrian B; Frey, Serita; Cabre, Anna; Ito, Takamitsu; Levine, Naomi M; Lønborg, Christian; Long, Matthew; Mauritz, Marguerite; Thomas, R Quinn; Stephens, Brandon M; Vanwalleghem, Tom; Zeng, Ning

    2016-01-01

    Organic matter (OM) plays a major role in both terrestrial and oceanic biogeochemical cycles. The amount of carbon stored in these systems is far greater than that of carbon dioxide (CO2 ) in the atmosphere, and annual fluxes of CO2 from these pools to the atmosphere exceed those from fossil fuel combustion. Understanding the processes that determine the fate of detrital material is important for predicting the effects that climate change will have on feedbacks to the global carbon cycle. However, Earth System Models (ESMs) typically utilize very simple formulations of processes affecting the mineralization and storage of detrital OM. Recent changes in our view of the nature of this material and the factors controlling its transformation have yet to find their way into models. In this review, we highlight the current understanding of the role and cycling of detrital OM in terrestrial and marine systems and examine how this pool of material is represented in ESMs. We include a discussion of the different mineralization pathways available as organic matter moves from soils, through inland waters to coastal systems and ultimately into open ocean environments. We argue that there is strong commonality between aspects of OM transformation in both terrestrial and marine systems and that our respective scientific communities would benefit from closer collaboration.

  10. Subcellular localization of RNA degrading proteins and protein complexes in prokaryotes.

    PubMed

    Evguenieva-Hackenberg, Elena; Roppelt, Verena; Lassek, Christian; Klug, Gabriele

    2011-01-01

    The archaeal exosome is a prokaryotic protein complex with RNA processing and degrading activities. Recently it was shown that the exosome is localized at the periphery of the cell in the thermoacidophilic archaeon Sulfolobus solfataricus. This localization is most likely mediated by the archaeal DnaG protein and depends on (direct or indirect) hydrophobic interactions with the membrane. A localization of RNA degrading proteins and protein complexes was also demonstrated in several bacteria. In bacteria a subcellular localization was also shown for substrates of these proteins and protein complexes, i.e. chromosomally encoded mRNAs and a small RNA. Thus, despite the missing compartmentalization, a spatial organization of RNA processing and degradation exists in prokaryotic cells. Recent data suggest that the spatial organization contributes to the temporal regulation of these processes.

  11. Casein kinase 1δ-dependent Wee1 protein degradation.

    PubMed

    Penas, Clara; Ramachandran, Vimal; Simanski, Scott; Lee, Choogon; Madoux, Franck; Rahaim, Ronald J; Chauhan, Ruchi; Barnaby, Omar; Schurer, Stephan; Hodder, Peter; Steen, Judith; Roush, William R; Ayad, Nagi G

    2014-07-04

    Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.

  12. Degradation kinetics and pathways of spirotetramat in different parts of spinach plant and in the soil.

    PubMed

    Chen, Xiaojun; Meng, Zhiyuan; Zhang, Yanyan; Gu, Haotian; Ren, Yajun; Lu, Chunliang

    2016-08-01

    Spirotetramat is a new pesticide against a broad spectrum of sucking insects and exhibits a unique property with a two-way systemicity. In order to formulate a scientific rationale for a reasonable spray dose and the safe interval period of 22.4 % spirotetramat suspension concentrate on controlling vegetable pests, we analyzed degradation dynamics and pathways of spirotetramat in different parts of spinach plant (leaf, stalk, and root) and in the soil. We conducted experimental trials under field conditions and adopted a simple and reliable method (dispersive solid phase extraction) combined with liquid chromatography-triple quadrupole tandem mass spectrometry to evaluate the dissipation rates of spirotetramat residue and its metabolites. The results showed that the spirotetramat was degraded into different metabolite residues in different parts of spinach plant (leaf, stalk, and root) and in the soil. Specifically, spirotetramat was degraded into B-keto, B-glu, and B-enol in the leaf; B-glu and B-enol in the stalk; and only B-enol in the root. In the soil where the plants grew, spirotetramat followed a completely different pathway compared to the plant and degraded into B-keto and B-mono. Regardless of different degradation pathways, the dissipation dynamic equations of spirotetramat in different parts of spinach plant and in the soil were all based on the first-order reaction dynamic equations. This work provides guidelines for the safe use of spirotetramat in spinach fields, which would help prevent potential health threats to consumers.

  13. Degradation pathway of malachite green in a novel dual-tank photoelectrochemical catalytic reactor.

    PubMed

    Diao, Zenghui; Li, Mingyu; Zeng, Fanyin; Song, Lin; Qiu, Rongliang

    2013-09-15

    A novel dual-tank photoelectrochemical catalytic reactor was designed to investigate the degradation pathway of malachite green. A thermally formed TiO₂/Ti thin film electrode was used as photoanode, graphite was used as cathode, and a saturated calomel electrode was employed as the reference electrode in the reactor. In the reactor, the anode and cathode tanks were connected by a cation exchange membrane. Results showed that the decolorization ratio of malachite green in the anode and cathode was 98.5 and 96.5% after 120 min, respectively. Malachite green in the two anode and cathode tanks was oxidized, achieving the bipolar double effect. Malachite green in both the anode and cathode tanks exhibited similar catalytic degradation pathways. The double bond of the malachite green molecule was attacked by strong oxidative hydroxyl radicals, after which the organic compound was degraded by the two pathways into 4,4-bis(dimethylamino) benzophenone, 4-(dimethylamino) benzophenone, 4-(dimethylamino) phenol, and other intermediate products. Eventually, malachite green was degraded into oxalic acid as a small molecular organic acid, which was degraded by processes such as demethylation, deamination, nitration, substitution, addition, and other reactions.

  14. Degradation of diclofenac by advanced oxidation and reduction processes: kinetic studies, degradation pathways and toxicity assessments.

    PubMed

    Yu, Hui; Nie, Er; Xu, Jun; Yan, Shuwen; Cooper, William J; Song, Weihua

    2013-04-01

    Many pharmaceutical compounds and metabolites are found in surface and ground waters suggesting their ineffective removal by conventional wastewater treatment technologies. Advanced oxidation/reduction processes (AO/RPs), which utilize free radical reactions to directly degrade chemical contaminants, are alternatives to traditional water treatment. This study reports the absolute rate constants for reaction of diclofenac sodium and model compound (2, 6-dichloraniline) with the two major AO/RP radicals: the hydroxyl radical (•OH) and hydrated electron (e(aq)(-)). The bimolecular reaction rate constants (M(-1) s(-1)) for diclofenac for •OH was (9.29 ± 0.11) × 10(9), and for e(-)(aq) was (1.53 ± 0.03) ×10(9). To provide a better understanding of the decomposition of the intermediate radicals produced by hydroxyl radical reactions, transient absorption spectra are observed from 1 - 250 μs. In addition, preliminary degradation mechanisms and major products were elucidated using (60)Co γ-irradiation and LC-MS. The toxicity of products was evaluated using luminescent bacteria. These data are required for both evaluating the potential use of AO/RPs for the destruction of these compounds and for studies of their fate and transport in surface waters where radical chemistry may be important in assessing their lifetime.

  15. Reconstructing metabolic pathways of hydrocarbon-degrading bacteria from the Deepwater Horizon oil spill.

    PubMed

    Dombrowski, Nina; Donaho, John A; Gutierrez, Tony; Seitz, Kiley W; Teske, Andreas P; Baker, Brett J

    2016-05-09

    The Deepwater Horizon blowout in the Gulf of Mexico in 2010, one of the largest marine oil spills(1), changed bacterial communities in the water column and sediment as they responded to complex hydrocarbon mixtures(2-4). Shifts in community composition have been correlated to the microbial degradation and use of hydrocarbons(2,5,6), but the full genetic potential and taxon-specific metabolisms of bacterial hydrocarbon degraders remain unresolved. Here, we have reconstructed draft genomes of marine bacteria enriched from sea surface and deep plume waters of the spill that assimilate alkane and polycyclic aromatic hydrocarbons during stable-isotope probing experiments, and we identify genes of hydrocarbon degradation pathways. Alkane degradation genes were ubiquitous in the assembled genomes. Marinobacter was enriched with n-hexadecane, and uncultured Alpha- and Gammaproteobacteria populations were enriched in the polycyclic-aromatic-hydrocarbon-degrading communities and contained a broad gene set for degrading phenanthrene and naphthalene. The repertoire of polycyclic aromatic hydrocarbon use varied among different bacterial taxa and the combined capabilities of the microbial community exceeded those of its individual components, indicating that the degradation of complex hydrocarbon mixtures requires the non-redundant capabilities of a complex oil-degrading community.

  16. Degradation of oxcarbazepine by UV-activated persulfate oxidation: kinetics, mechanisms, and pathways.

    PubMed

    Bu, Lingjun; Zhou, Shiqing; Shi, Zhou; Deng, Lin; Li, Guangchao; Yi, Qihang; Gao, Naiyun

    2016-02-01

    The degradation kinetics and mechanism of the antiepileptic drug oxcarbazepine (OXC) by UV-activated persulfate oxidation were investigated in this study. Results showed that UV/persulfate (UV/PS) process appeared to be more effective in degrading OXC than UV or PS alone. The OXC degradation exhibited a pseudo-first order kinetics pattern and the degradation rate constants (k obs) were affected by initial OXC concentration, PS dosage, initial pH, and humic acid concentration to different degrees. It was found that low initial OXC concentration, high persulfate dosage, and initial pH enhanced the OXC degradation. Additionally, the presence of humic acid in the solution could greatly inhibit the degradation of OXC. Moreover, hydroxyl radical (OH•) and sulfate radical (SO4 (-)••) were identified to be responsible for OXC degradation and SO4 (-)• made the predominant contribution in this study. Finally, major intermediate products were identified and a preliminary degradation pathway was proposed. Results demonstrated that UV/PS system is a potential technology to control the water pollution caused by emerging contaminants such as OXC.

  17. Lactational evaluation of protein supplements of varying ruminal degradabilities.

    PubMed

    Henson, J E; Schingoethe, D J; Maiga, H A

    1997-02-01

    Twelve lactating Holstein cows (9 multiparous and 3 primiparous) were used in a replicated 3 x 3 Latin square design with three periods of 4 wk each to evaluate diets containing three protein supplements that varied in ruminally undegradable protein and amino acid (AA) composition. Diets contained either 44% crude protein (CP) solvent-extracted soybean meal, expeller (mechanically extracted) soybean meal, or a blend of animal and vegetable proteins as the protein supplement. The animal and vegetable blend consisted of equal portions of protein from blood meal, corn gluten meal, meat and bone meal, and soybean meal. All diets contained 33.3% alfalfa haylage, 16.7% corn silage, and 50% of the respective concentrate mix (dry matter basis). Diets contained 17.4, 17.8, and 17.8% CP and 34, 45, and 45% of CP as ruminally undegradable protein, respectively. Dry matter intake, milk production and composition, and body weight were similar among treatments. Uptakes of AA by the mammary gland were similar among treatments. The apparent first-limiting AA for each diet was likely Met, but Lys and Phe were also potentially limiting. Varying degrees of protein degradability and AA composition within the range of this study did not affect lactational responses, indicating that all of these protein supplements were adequate to support milk production.

  18. Kinetic models and pathways of ronidazole degradation by chlorination, UV irradiation and UV/chlorine processes.

    PubMed

    Qin, Lang; Lin, Yi-Li; Xu, Bin; Hu, Chen-Yan; Tian, Fu-Xiang; Zhang, Tian-Yang; Zhu, Wen-Qian; Huang, He; Gao, Nai-Yun

    2014-11-15

    Degradation kinetics and pathways of ronidazole (RNZ) by chlorination (Cl2), UV irradiation and combined UV/chlorine processes were investigated in this paper. The degradation kinetics of RNZ chlorination followed a second-order behavior with the rate constants calculated as (2.13 ± 0.15) × 10(2) M(-2) s(-1), (0.82 ± 0.52) × 10(-2) M(-1) s(-1) and (2.06 ± 0.09) × 10(-1) M(-1) s(-1) for the acid-catalyzed reaction, as well as the reactions of RNZ with HOCl and OCl(-), respectively. Although UV irradiation degraded RNZ more effectively than chlorination did, very low quantum yield of RNZ at 254 nm was obtained as 1.02 × 10(-3) mol E(-1). RNZ could be efficiently degraded and mineralized in the UV/chlorine process due to the generation of hydroxyl radicals. The second-order rate constant between RNZ and hydroxyl radical was determined as (2.92 ± 0.05) × 10(9) M(-1) s(-1). The degradation intermediates of RNZ during the three processes were identified with Ultra Performance Liquid Chromatography - Electrospray Ionization - mass spectrometry and the degradation pathways were then proposed. Moreover, the variation of chloropicrin (TCNM) and chloroform (CF) formation after the three processes were further evaluated. Enhanced formation of CF and TCNM precursors during UV/chlorine process deserves extensive attention in drinking water treatment.

  19. A novel pathway for nicotine degradation by Aspergillus oryzae 112822 isolated from tobacco leaves.

    PubMed

    Meng, Xiang Jing; Lu, Li Li; Gu, Guo Feng; Xiao, Min

    2010-09-01

    An efficient nicotine-degrading fungus was isolated from tobacco leaves and identified as Aspergillus oryzae 112822 based on morphological characteristics and sequence analysis of 18S rDNA, 5.8S rDNA and the internal transcribed spacer (5.8S-ITS region). When the strain was cultured in a medium with tobacco leaf extract for 40 h, the maximum amount of cell growth was 3.6 g l(-1) and nicotine degradation was 2.19 g l(-1). The intermediates of nicotine degradation by resting cells were isolated by preparative TLC or semi-preparative HPLC, and identified by TLC, MS, NMR, Fourier-transform (FT)-IR and GC-MS analysis. The pathway for nicotine degradation in A. oryzae 112822 was proposed to be from nicotine to 2,3-dihydroxypyridine through the intermediates nornicotine, myosmine, N-methylnicotinamide and 2-hydroxy-N-methylnicotinamide. The ring of 2,3-dihydroxypyridine was opened between the 2- and 3-hydroxy positions to yield succinic acid. N-methylnicotinamide and 2,3-dihydroxypyridine were satisfactorily verified as metabolites of nicotine degradation. This is the first elucidation of a pathway for nicotine degradation in fungi.

  20. Attenuation of glucocorticoid signaling through targeted degradation of p300 via the 26S proteasome pathway.

    PubMed

    Li, Qiao; Su, Anna; Chen, Jihong; Lefebvre, Yvonne A; Haché, Robert J G

    2002-12-01

    The effects of acetylation on gene expression are complex, with changes in chromatin accessibility intermingled with direct effects on transcriptional regulators. For the nuclear receptors, both positive and negative effects of acetylation on specific gene transcription have been observed. We report that p300 and steroid receptor coactivator 1 interact transiently with the glucocorticoid receptor and that the acetyltransferase activity of p300 makes an important contribution to glucocorticoid receptor-mediated transcription. Treatment of cells with the deacetylase inhibitor, sodium butyrate, inhibited steroid-induced transcription and altered the transient association of glucocorticoid receptor with p300 and steroid receptor coactivator 1. Additionally, sustained sodium butyrate treatment induced the degradation of p300 through the 26S proteasome pathway. Treatment with the proteasome inhibitor MG132 restored both the level of p300 protein and the transcriptional response to steroid over 20 h of treatment. These results reveal new levels for the regulatory control of gene expression by acetylation and suggest feedback control on p300 activity.

  1. Evolutionary, computational, and biochemical studies of the salicylaldehyde dehydrogenases in the naphthalene degradation pathway

    PubMed Central

    Jia, Baolei; Jia, Xiaomeng; Hyun Kim, Kyung; Ji Pu, Zhong; Kang, Myung-Suk; Ok Jeon, Che

    2017-01-01

    Salicylaldehyde (SAL) dehydrogenase (SALD) is responsible for the oxidation of SAL to salicylate using nicotinamide adenine dinucleotide (NAD+) as a cofactor in the naphthalene degradation pathway. We report the use of a protein sequence similarity network to make functional inferences about SALDs. Network and phylogenetic analyses indicated that SALDs and the homologues are present in bacteria and fungi. The key residues in SALDs were analyzed by evolutionary methods and a molecular simulation analysis. The results showed that the catalytic residue is most highly conserved, followed by the residues binding NAD+ and then the residues binding SAL. A molecular simulation analysis demonstrated the binding energies of the amino acids to NAD+ and/or SAL and showed that a conformational change is induced by binding. A SALD from Alteromonas naphthalenivorans (SALDan) that undergoes trimeric oligomerization was characterized enzymatically. The results showed that SALDan could catalyze the oxidation of a variety of aromatic aldehydes. Site-directed mutagenesis of selected residues binding NAD+ and/or SAL affected the enzyme’s catalytic efficiency, but did not eliminate catalysis. Finally, the relationships among the evolution, catalytic mechanism, and functions of SALD are discussed. Taken together, this study provides an expanded understanding of the evolution, functions, and catalytic mechanism of SALD. PMID:28233868

  2. Myostatin induces p300 degradation to silence cyclin D1 expression through the PI3K/PTEN/Akt pathway.

    PubMed

    Ji, Ming; Zhang, Qiang; Ye, Jianwei; Wang, Xueyan; Yang, Wei; Zhu, Dahai

    2008-08-01

    Myostatin is a negative regulator of skeletal muscle growth and affects numerous genes expression involved in cell proliferation, differentiation and metabolism. However, the molecular mechanisms underlying myostatin-regulated genes expression remain to be elucidated. In this study, we showed that myostatin blocked the recruitment of p300 to the cyclin D1 promoter, resulting in the silence of cyclin D1 expression. Our data further demonstrated that myostatin decreased the protein level of p300 by inducing p300 degradation via the ubiquitin-proteasome system. In addition, we provided experimental evidence to show that myostatin-induced p300 degradation was mediated by the phosphatidylinositol 3-kinase/PTEN/Akt signaling pathway and this could be antagonized by IGF-1 or insulin. Results presented in this study uncovered an epigenetic control of genes expression in response to myostatin.

  3. Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum.

    PubMed

    Kitamura, Takuya; Seki, Naoya; Kihara, Akio

    2017-03-28

    Although normal fatty acids (FAs) are degraded via β-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2-deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

  4. Cooperative catabolic pathways within an atrazine-degrading enrichment culture isolated from soil.

    PubMed

    Smith, Daniel; Alvey, Sam; Crowley, David E

    2005-07-01

    Atrazine degradation previously has been shown to be carried out by individual bacterial species or by relatively simple consortia that have been isolated using enrichment cultures. Here, the degradative pathway for atrazine was examined for a complex 8-membered enrichment culture. The species composition of the culture was determined by PCR-DGGE. The bacterial species included Agrobacterium tumefaciens, Caulobacter crescentus, Pseudomonas putida, Sphingomonas yaniokuyae, Nocardia sp., Rhizobium sp., Flavobacterium oryzihabitans, and Variovorax paradoxus. All of the isolates were screened for the presence of known genes that function for atrazine degradation including atzA,-B,-C,-D,-E,-F and trzD,-N. Dechlorination of atrazine, which was obligatory for complete mineralization, was carried out exclusively by Nocardia sp., which contained the trzN gene. Following dechlorination, the resulting product, hydroxyatrazine was further degraded via two separate pathways. In one pathway Nocardia converted hydroxyatrazine to N-ethylammelide via an unidentified gene product. In the second pathway, hydroxyatrazine generated by Nocardia sp. was hydrolyzed to N-isopropylammelide by Rhizobium sp., which contained the atzB gene. Each member of the enrichment culture contained atzC, which is responsible for ring cleavage, but none of the isolates carried the atzD,-E, or -F genes. Each member further contained either trzD or exhibited urease activity. The enrichment culture was destabilized by loss of Nocardia sp. when grown on ethylamine, ethylammelide, and cyanuric acid, after which the consortium was no longer able to degrade atrazine. The analysis of this enrichment culture highlights the broad level bacterial community interactions that may be involved in atrazine degradation in nature.

  5. Integrated protein quality-control pathways regulate free α-globin in murine β-thalassemia

    PubMed Central

    Khandros, Eugene; Thom, Christopher S.; D'Souza, Janine

    2012-01-01

    Cells remove unstable polypeptides through protein quality-control (PQC) pathways such as ubiquitin-mediated proteolysis and autophagy. In the present study, we investigated how these pathways are used in β-thalassemia, a common hemoglobinopathy in which β-globin gene mutations cause the accumulation and precipitation of cytotoxic α-globin subunits. In β-thalassemic erythrocyte precursors, free α-globin was polyubiquitinated and degraded by the proteasome. These cells exhibited enhanced proteasome activity, and transcriptional profiling revealed coordinated induction of most proteasome subunits that was mediated by the stress-response transcription factor Nrf1. In isolated thalassemic cells, short-term proteasome inhibition blocked the degradation of free α-globin. In contrast, prolonged in vivo treatment of β-thalassemic mice with the proteasome inhibitor bortezomib did not enhance the accumulation of free α-globin. Rather, systemic proteasome inhibition activated compensatory proteotoxic stress-response mechanisms, including autophagy, which cooperated with ubiquitin-mediated proteolysis to degrade free α-globin in erythroid cells. Our findings show that multiple interregulated PQC responses degrade excess α-globin. Therefore, β-thalassemia fits into the broader framework of protein-aggregation disorders that use PQC pathways as cell-protective mechanisms. PMID:22427201

  6. Bacterial community structure and predicted alginate metabolic pathway in an alginate-degrading bacterial consortium.

    PubMed

    Kita, Akihisa; Miura, Toyokazu; Kawata, Satoshi; Yamaguchi, Takeshi; Okamura, Yoshiko; Aki, Tsunehiro; Matsumura, Yukihiko; Tajima, Takahisa; Kato, Junichi; Nishio, Naomichi; Nakashimada, Yutaka

    2016-03-01

    Methane fermentation is one of the effective approaches for utilization of brown algae; however, this process is limited by the microbial capability to degrade alginate, a main polysaccharide found in these algae. Despite its potential, little is known about anaerobic microbial degradation of alginate. Here we constructed a bacterial consortium able to anaerobically degrade alginate. Taxonomic classification of 16S rRNA gene, based on high-throughput sequencing data, revealed that this consortium included two dominant strains, designated HUA-1 and HUA-2; these strains were related to Clostridiaceae bacterium SK082 (99%) and Dysgonomonas capnocytophagoides (95%), respectively. Alginate lyase activity and metagenomic analyses, based on high-throughput sequencing data, revealed that this bacterial consortium possessed putative genes related to a predicted alginate metabolic pathway. However, HUA-1 and 2 did not grow on agar medium with alginate by using roll-tube method, suggesting the existence of bacterial interactions like symbiosis for anaerobic alginate degradation.

  7. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system

    PubMed Central

    Guharoy, Mainak; Bhowmick, Pallab; Sallam, Mohamed; Tompa, Peter

    2016-01-01

    Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation. PMID:26732515

  8. 1,3-Dinitrobenzene reductive degradation by alkaline ascorbic acid - Reaction mechanisms, degradation pathways and reagent optimization.

    PubMed

    Ciou, Chiya; Liang, Chenju

    2017-01-01

    Nitro-aromatic compounds (NACs) such as 1,3-dinitrobenzene (1,3-DNB) contain the nitrogroup (-NO2), in which the N with a +III oxidation state accepts electrons. Water soluble ascorbic acid (AsA) at elevated pH produces electron transfer and governs the electron-donating pathway. The influence of the NaOH/AsA molar ratio on the degradation of 1,3-DNB was investigated. Using 0.21-2 M NaOH and 20-100 mM AsA, nearly complete 1,3-DNB removals (90-100%) were achieved within 0.5 h. On the basis of intermediates identified using GC/MS, the reduction pathways of 1,3-DNB can be categorized into step-by-step electron transfer, and condensation routes. A higher NaOH/AsA molar ratio would result in relatively higher AsA decomposition, promote the condensation route into the formation of azo- and azoxy-compounds, and ultimately reduce 1,3-DNB to 1,3-phenylenediamine. Contaminated soil flushing using 500 mM NaOH/100 mM AsA revealed that 1,3-DNB was completely degraded within 2 h. Based on these test results, the alkaline AsA treatment method is a potential remediation process for NACs contaminated soils.

  9. Degradation kinetics and pathway of phenol by Pseudomonas and Bacillus species.

    PubMed

    Hasan, Syed Adnan; Jabeen, Suraiya

    2015-01-02

    This article elucidates that strain Pseudomonas aeruginosa (IES-Ps-1) is a versatile toxic organic compound degrader. With the degradation of malathion and cypermethrin (studied by other researchers previously), this strain was able to degrade phenol. Two other indigenous soil flora (i.e., Pseudomonas sp. (IES-S) and Bacillus subtilis (IES-B)) were also found to be potential phenol degraders. Phenol was degraded with Monod kinetics during growth in nutrient broth and mineral salts medium. Before entering into the growth inhibition phase, strains IES-Ps-1, IES-S and IES-B could tolerate up to 400, 700 and 500 mg/L phenol, respectively, when contained in nutrient broth. However, according to the Luong-Levenspiel model, the growth of strains IES-Ps-1, IES-S and IES-B would cease at 2000, 2174 and 2190 mg/L phenol, respectively. Strain IES-Ps-1 degraded 700, 900 and 1050 mg/L phenol contained in mineral salts medium with the specific rates of 0.034, 0.075 and 0.021 h(-1), respectively. All these strains grew by making clusters when exposed to phenol in order to prevent damages due to high substrate concentration. These strains transformed phenol into catechol, which was then degraded via ortho-cleavage pathway.

  10. Degradation kinetics and pathway of phenol by Pseudomonas and Bacillus species

    PubMed Central

    Hasan, Syed Adnan; Jabeen, Suraiya

    2015-01-01

    This article elucidates that strain Pseudomonas aeruginosa (IES-Ps-1) is a versatile toxic organic compound degrader. With the degradation of malathion and cypermethrin (studied by other researchers previously), this strain was able to degrade phenol. Two other indigenous soil flora (i.e., Pseudomonas sp. (IES-S) and Bacillus subtilis (IES-B)) were also found to be potential phenol degraders. Phenol was degraded with Monod kinetics during growth in nutrient broth and mineral salts medium. Before entering into the growth inhibition phase, strains IES-Ps-1, IES-S and IES-B could tolerate up to 400, 700 and 500 mg/L phenol, respectively, when contained in nutrient broth. However, according to the Luong–Levenspiel model, the growth of strains IES-Ps-1, IES-S and IES-B would cease at 2000, 2174 and 2190 mg/L phenol, respectively. Strain IES-Ps-1 degraded 700, 900 and 1050 mg/L phenol contained in mineral salts medium with the specific rates of 0.034, 0.075 and 0.021 h−1, respectively. All these strains grew by making clusters when exposed to phenol in order to prevent damages due to high substrate concentration. These strains transformed phenol into catechol, which was then degraded via ortho-cleavage pathway. PMID:26740787

  11. Degenerin channel activation causes caspase‐mediated protein degradation and mitochondrial dysfunction in adult C. elegans muscle

    PubMed Central

    Gaffney, Christopher J.; Shephard, Freya; Chu, Jeff; Baillie, David L.; Rose, Ann; Constantin‐Teodosiu, Dumitru; Greenhaff, Paul L.

    2015-01-01

    Abstract Background Declines in skeletal muscle structure and function are found in various clinical populations, but the intramuscular proteolytic pathways that govern declines in these individuals remain relatively poorly understood. The nematode Caenorhabditis elegans has been developed into a model for identifying and understanding these pathways. Recently, it was reported that UNC‐105/degenerin channel activation produced muscle protein degradation via an unknown mechanism. Methods Generation of transgenic and double mutant C. elegans, RNAi, and drug treatments were utilized to assess molecular events governing protein degradation. Western blots were used to measure protein content. Cationic dyes and adenosine triphosphate (ATP) production assays were utilized to measure mitochondrial function. Results unc‐105 gain‐of‐function mutants display aberrant muscle protein degradation and a movement defect; both are reduced in intragenic revertants and in let‐2 mutants that gate the hyperactive UNC‐105 channel. Degradation is not suppressed by interventions suppressing proteasome‐mediated, autophagy‐mediated, or calpain‐mediated degradation nor by suppressors of degenerin‐induced neurodegeneration. Protein degradation, but not the movement defect, is decreased by treatment with caspase inhibitors or RNAi against ced‐3 or ced‐4. Adult unc‐105 muscles display a time‐dependent fragmentation of the mitochondrial reticulum that is associated with impaired mitochondrial membrane potential and that correlates with decreased rates of maximal ATP production. Reduced levels of CED‐4, which is sufficient to activate CED‐3 in vitro, are observed in unc‐105 mitochondrial isolations. Conclusions Constitutive cationic influx into muscle appears to cause caspase degradation of cytosolic proteins as the result of mitochondrial dysfunction, which may be relevant to ageing and sarcopenia. PMID:27493871

  12. [Lipid- and protein-degrading processes during the maturation of ham].

    PubMed

    López Bote, C; Córdoba, J J; Antequera, T

    1993-02-01

    In the present work we review the main degradative pathways for lipids and proteins along the ripening of dry cured hams, with special emphasis on Iberian pig hams. Maximum proteolytic activity is found around the first stages (salting) and specially at the dryer. Lipolytic activity seems to be also higher in this stage. During the steps that follow the post-salting period the oxidation seems to be activated. The products from proteolytic and lipolytic processes might react among each other during the final steps in the cellar.

  13. Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis.

    PubMed

    Briz, Victor; Hsu, Yu-Tien; Li, Yi; Lee, Erin; Bi, Xiaoning; Baudry, Michel

    2013-03-06

    Memory consolidation has been suggested to be protein synthesis dependent. Previous data indicate that BDNF-induced dendritic protein synthesis is a key event in memory formation through activation of the mammalian target of rapamycin (mTOR) pathway. BDNF also activates calpain, a calcium-dependent cysteine protease, which has been shown to play a critical role in learning and memory. This study was therefore directed at testing the hypothesis that calpain activity is required for BDNF-stimulated local protein synthesis, and at identifying the underlying molecular mechanism. In rat hippocampal slices, cortical synaptoneurosomes, and cultured neurons, BDNF-induced mTOR pathway activation and protein translation were blocked by calpain inhibition. BDNF treatment rapidly reduced levels of hamartin and tuberin, negative regulators of mTOR, in a calpain-dependent manner. Treatment of brain homogenates with purified calpain-1 and calpain-2 truncated both proteins. BDNF treatment increased phosphorylation of both Akt and ERK, but only the effect on Akt was blocked by calpain inhibition. Levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatase that inactivates Akt, were decreased following BDNF treatment, and calpain inhibition reversed this effect. Calpain-2, but not calpain-1, treatment of brain homogenates resulted in PTEN degradation. In cultured cortical neurons, knockdown of calpain-2, but not calpain-1, by small interfering RNA completely suppressed the effect of BDNF on mTOR activation. Our results reveal a critical role for calpain-2 in BDNF-induced mTOR signaling and dendritic protein synthesis via PTEN, hamartin, and tuberin degradation. This mechanism therefore provides a link between proteolysis and protein synthesis that might contribute to synaptic plasticity.

  14. Design principles of a universal protein degradation machine.

    PubMed

    Matyskiela, Mary E; Martin, Andreas

    2013-01-23

    The 26S proteasome is a 2.5-MDa, 32-subunit ATP-dependent protease that is responsible for the degradation of ubiquitinated protein targets in all eukaryotic cells. This proteolytic machine consists of a barrel-shaped peptidase capped by a large regulatory particle, which contains a heterohexameric AAA+ unfoldase as well as several structural modules of previously unknown function. Recent electron microscopy (EM) studies have allowed major breakthroughs in understanding the architecture of the regulatory particle, revealing that the additional modules provide a structural framework to position critical, ubiquitin-interacting subunits and thus allow the 26S proteasome to function as a universal degradation machine for a wide variety of protein substrates. The EM studies have also uncovered surprising asymmetries in the spatial arrangement of proteasome subunits, yet the functional significance of these architectural features remains unclear. This review will summarize the recent findings on 26S proteasome structure and discuss the mechanistic implications for substrate binding, deubiquitination, unfolding, and degradation.

  15. Degradation of ibuprofen by hydrodynamic cavitation: Reaction pathways and effect of operational parameters.

    PubMed

    Musmarra, Dino; Prisciandaro, Marina; Capocelli, Mauro; Karatza, Despina; Iovino, Pasquale; Canzano, Silvana; Lancia, Amedeo

    2016-03-01

    Ibuprofen (IBP) is an anti-inflammatory drug whose residues can be found worldwide in natural water bodies resulting in harmful effects to aquatic species even at low concentrations. This paper deals with the degradation of IBP in water by hydrodynamic cavitation in a convergent-divergent nozzle. Over 60% of ibuprofen was degraded in 60 min with an electrical energy per order (EEO) of 10.77 kWh m(-3) at an initial concentration of 200 μg L(-1) and a relative inlet pressure pin=0.35 MPa. Five intermediates generated from different hydroxylation reactions were identified; the potential mechanisms of degradation were sketched and discussed. The reaction pathways recognized are in line with the relevant literature, both experimental and theoretical. By varying the pressure upstream the constriction, different degradation rates were observed. This effect was discussed according to a numerical simulation of the hydroxyl radical production identifying a clear correspondence between the maximum kinetic constant kOH and the maximum calculated OH production. Furthermore, in the investigated experimental conditions, the pH parameter was found not to affect the extent of degradation; this peculiar feature agrees with a recently published kinetic insight and has been explained in the light of the intermediates of the different reaction pathways.

  16. Metagenomic identification of bacterioplankton taxa and pathways involved in microcystin degradation in lake erie.

    PubMed

    Mou, Xiaozhen; Lu, Xinxin; Jacob, Jisha; Sun, Shulei; Heath, Robert

    2013-01-01

    Cyanobacterial harmful blooms (CyanoHABs) that produce microcystins are appearing in an increasing number of freshwater ecosystems worldwide, damaging quality of water for use by human and aquatic life. Heterotrophic bacteria assemblages are thought to be important in transforming and detoxifying microcystins in natural environments. However, little is known about their taxonomic composition or pathways involved in the process. To address this knowledge gap, we compared the metagenomes of Lake Erie free-living bacterioplankton assemblages in laboratory microcosms amended with microcystins relative to unamended controls. A diverse array of bacterial phyla were responsive to elevated supply of microcystins, including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria of the alpha, beta, gamma, delta and epsilon subdivisions and Verrucomicrobia. At more detailed taxonomic levels, Methylophilales (mainly in genus Methylotenera) and Burkholderiales (mainly in genera Bordetella, Burkholderia, Cupriavidus, Polaromonas, Ralstonia, Polynucleobacter and Variovorax) of Betaproteobacteria were suggested to be more important in microcystin degradation than Sphingomonadales of Alphaproteobacteria. The latter taxa were previously thought to be major microcystin degraders. Homologs to known microcystin-degrading genes (mlr) were not overrepresented in microcystin-amended metagenomes, indicating that Lake Erie bacterioplankton might employ alternative genes and/or pathways in microcystin degradation. Genes for xenobiotic metabolism were overrepresented in microcystin-amended microcosms, suggesting they are important in bacterial degradation of microcystin, a phenomenon that has been identified previously only in eukaryotic systems.

  17. Degradation kinetics and pathways of three calcium channel blockers under UV irradiation.

    PubMed

    Zhu, Bing; Zonja, Bozo; Gonzalez, Oscar; Sans, Carme; Pérez, Sandra; Barceló, Damia; Esplugas, Santiago; Xu, Ke; Qiang, Zhimin

    2015-12-01

    Calcium channel blockers (CCBs) are a group of pharmaceuticals widely prescribed to lower blood pressure and treat heart diseases. They have been frequently detected in wastewater treatment plant (WWTP) effluents and downstream river waters, thus inducing a potential risk to aquatic ecosystems. However, little is known about the behavior and fate of CCBs under UV irradiation, which has been adopted as a primary disinfection method for WWTP effluents. This study investigated the degradation kinetics and pathways of three commonly-used CCBs, including amlodipine (AML), diltiazem (DIL), and verapamil (VER), under UV (254 nm) irradiation. The chemical structures of transformation byproducts (TBPs) were first identified to assess the potential ecological hazards. On that basis, a generic solid-phase extraction method, which simultaneously used four different cartridges, was adopted to extract and enrich the TBPs. Thereafter, the photo-degradation of target CCBs was performed under UV fluences typical for WWTP effluent disinfection. The degradation of all three CCBs conformed to the pseudo-first-order kinetics, with rate constants of 0.031, 0.044 and 0.011 min(-1) for AML, DIL and VER, respectively. By comparing the MS(2) fragments and the evolution (i.e., formation or decay) trends of identified TBPs, the degradation pathways were proposed. In the WWTP effluent, although the target CCBs could be degraded, several TBPs still contained the functional pharmacophores and reached peak concentrations under UV fluences of 40-100 mJ cm(-2).

  18. Catalytic thermolysis in treating Cibacron Blue in aqueous solution: Kinetics and degradation pathway.

    PubMed

    Su, Claire Xin-Hui; Teng, Tjoon-Tow; Wong, Yee-Shian; Morad, Norhashimah; Rafatullah, Mohd

    2016-03-01

    A thermal degradation pathway of the decolourisation of Reactive Cibacron Blue F3GA (RCB) in aqueous solution through catalytic thermolysis is established. Catalytic thermolysis is suitable for the removal of dyes from wastewater as it breaks down the complex dye molecules instead of only transferring them into another phase. RCB is a reactive dye that consists of three main groups, namely anthraquinone, benzene and triazine groups. Through catalytic thermolysis, the bonds that hold the three groups together were effectively broken and at the same time, the complex molecules degraded to form simple molecules of lower molecular weight. The degradation pathway and products were characterized and determined through UV-Vis, FT-IR and GCMS analysis. RCB dye molecule was successfully broken down into simpler molecules, namely, benzene derivatives, amines and triazine. The addition of copper sulphate, CuSO4, as a catalyst, hastens the thermal degradation of RCB by aiding in the breakdown of large, complex molecules. At pH 2 and catalyst mass loading of 5 g/L, an optimum colour removal of 66.14% was observed. The degradation rate of RCB is well explained by first order kinetics model.

  19. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity.

    PubMed

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-12-17

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the β-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of β-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish.

  20. A Polyomic Approach To Elucidate the Fluoranthene-Degradative Pathway in Mycobacterium vanbaalenii PYR-1▿ †

    PubMed Central

    Kweon, Ohgew; Kim, Seong-Jae; Jones, Richard C.; Freeman, James P.; Adjei, Michael D.; Edmondson, Ricky D.; Cerniglia, Carl E.

    2007-01-01

    Mycobacterium vanbaalenii PYR-1 is capable of degrading a wide range of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs), including fluoranthene. We used a combination of metabolomic, genomic, and proteomic technologies to investigate fluoranthene degradation in this strain. Thirty-seven fluoranthene metabolites including potential isomers were isolated from the culture medium and analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and UV-visible absorption. Total proteins were separated by one-dimensional gel and analyzed by liquid chromatography-tandem mass spectrometry in conjunction with the M. vanbaalenii PYR-1 genome sequence (http://jgi.doe.gov), which resulted in the identification of 1,122 proteins. Among them, 53 enzymes were determined to be likely involved in fluoranthene degradation. We integrated the metabolic information with the genomic and proteomic results and proposed pathways for the degradation of fluoranthene. According to our hypothesis, the oxidation of fluoranthene is initiated by dioxygenation at the C-1,2, C-2,3, and C-7,8 positions. The C-1,2 and C-2,3 dioxygenation routes degrade fluoranthene via fluorene-type metabolites, whereas the C-7,8 routes oxidize fluoranthene via acenaphthylene-type metabolites. The major site of dioxygenation is the C-2,3 dioxygenation route, which consists of 18 enzymatic steps via 9-fluorenone-1-carboxylic acid and phthalate with the initial ring-hydroxylating oxygenase, NidA3B3, oxidizing fluoranthene to fluoranthene cis-2,3-dihydrodiol. Nonspecific monooxygenation of fluoranthene with subsequent O methylation of dihydroxyfluoranthene also occurs as a detoxification reaction. PMID:17449607

  1. Pathway illuminated: visualizing protein kinase C signaling.

    PubMed

    Violin, Jonathan D; Newton, Alexandra C

    2003-12-01

    Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals.

  2. Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

    PubMed

    Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

    2016-08-01

    Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.

  3. The Nuclear Factor-kB Pathway Regulates Cytochrome P450 3A4 Protein Stability

    SciTech Connect

    Zangar, Richard C.; Bollinger, Nikki; Verma, Seema; Karin, Norm J.; Lu, Yi

    2008-06-01

    We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 is degraded by the proteasome, it seems likely that there is another protein that is susceptible to proteasomal degradation that regulates CYP3A4 expression. In this study, we evaluate whether the nuclear factor kappa B (NF-kB) pathway is involved in that process. Our model system uses an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, we found that inhibition of the proteasome with MG132 suppresses CYP3A4. Consistent with reports that proteasome inhibition suppresses the NF-kB pathway, we also observe a suppression of inhibitory kB kinase protein levels after treatment with MG132. Treatment of the HepG2 cells with NK-kB Activation Inhibitor also suppresses CYP3A4 proteins levels. In contrast, inhibition of either the proteasome or NF-kB pathways increases CYP3A4 mRNA levels. When the HepG2 cells are treated with cycloheximide, a general inhibitor of translation, the loss of CYP3A4 protein is accelerated by co-treatment with an NF-kB Activation Inhibitor. These results indicate that NF-kB activity regulates CYP3A4 protein stability and suggest that the NF-kB pathway is responsible for the decrease in CYP3A4 protein levels that results from the inhibition of proteasomal activity.

  4. Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6- Dinitrotoluene.

    DTIC Science & Technology

    2000-01-01

    of 3M4NC, 9.8 t±mol of sodium phosphate (pH 7.0), and cell extract (0.1 been designated Alcaligenes sp. They are represented by strain to 0.5 mg of...protein) in a final volume of 1 ml. The molar extinction coefficients JS867 ( Alcaligenes denitrificans Biolog cluster) and strain JS871 of compounds X...cepacia Pseudomonas sp. 1; 1992 2,4 - + + PR7 Burkholderia cepacia Pseudomonas sp. 1; 1992 2,4 - + + JS850 Burkholderia cepacia None 3; 1995 2,6

  5. Modulation of signaling pathways by RNA virus capsid proteins.

    PubMed

    Urbanowski, Matthew D; Ilkow, Carolina S; Hobman, Tom C

    2008-07-01

    Capsid proteins are structural components of virus particles. They are nucleic acid-binding proteins whose main recognized function is to package viral genomes into protective structures called nucleocapsids. Research over the last 10 years indicates that in addition to their role as genome guardians, viral capsid proteins modulate host cell signaling networks. Disruption or alteration of intracellular signaling pathways by viral capsids may benefit replication of the virus by affecting innate immunity and in some cases, may underlie disease progression. In this review, we describe how the capsid proteins from medically relevant RNA viruses interact with host cell signaling pathways.

  6. Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes.

    PubMed

    Yu, Jun; Lan, Xiang; Chen, Xia; Yu, Chao; Xu, Yiwen; Liu, Yujuan; Xu, Lingna; Fan, Heng-Yu; Tong, Chao

    2016-08-15

    The homeostasis of self-renewal and differentiation in stem cells is controlled by intrinsic signals and their niche. We conducted a large-scale RNA interference (RNAi) screen in Drosophila testes and identified 221 genes required for germline stem cell (GSC) maintenance or differentiation. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in the protein degradation pathway in cyst cells leads to testis tumors consisting of overproliferated germ cells. Importantly, in the Cullin 4-RING E3 ubiquitin ligase (CRL4) complex, we identified multiple proteins that are crucial to GSC self-renewal: pic/DDB1, a CRL4 linker protein, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice.

  7. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    PubMed Central

    Li, Shan; Shu, Bing; Zhang, Yanquan; Li, Jia; Guo, Junwei; Wang, Yinyin; Ren, Fangli; Xiao, Guozhi; Chang, Zhijie; Chen, Di

    2014-01-01

    Objective Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling. Methods The bone phenotype of Chip−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays. Results In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription. Conclusion CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases. PMID:24578159

  8. The relationship between protein synthesis and protein degradation in object recognition memory.

    PubMed

    Furini, Cristiane R G; Myskiw, Jociane de C; Schmidt, Bianca E; Zinn, Carolina G; Peixoto, Patricia B; Pereira, Luiza D; Izquierdo, Ivan

    2015-11-01

    For decades there has been a consensus that de novo protein synthesis is necessary for long-term memory. A second round of protein synthesis has been described for both extinction and reconsolidation following an unreinforced test session. Recently, it was shown that consolidation and reconsolidation depend not only on protein synthesis but also on protein degradation by the ubiquitin-proteasome system (UPS), a major mechanism responsible for protein turnover. However, the involvement of UPS on consolidation and reconsolidation of object recognition memory remains unknown. Here we investigate in the CA1 region of the dorsal hippocampus the involvement of UPS-mediated protein degradation in consolidation and reconsolidation of object recognition memory. Animals with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus, were exposed to an object recognition task. The UPS inhibitor β-Lactacystin did not affect the consolidation and the reconsolidation of object recognition memory at doses known to affect other forms of memory (inhibitory avoidance, spatial learning in a water maze) while the protein synthesis inhibitor anisomycin impaired the consolidation and the reconsolidation of the object recognition memory. However, β-Lactacystin was able to reverse the impairment caused by anisomycin on the reconsolidation process in the CA1 region of the hippocampus. Therefore, it is possible to postulate a direct link between protein degradation and protein synthesis during the reconsolidation of the object recognition memory.

  9. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

    PubMed

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-15

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.

  10. New metabolic pathway for degradation of 2-nitrobenzoate by Arthrobacter sp. SPG

    PubMed Central

    Arora, Pankaj K.; Sharma, Ashutosh

    2015-01-01

    Arthrobacter sp. SPG utilized 2-nitrobenzoate as its sole source of carbon and energy and degraded it with accumulation of stoichiometric amounts of nitrite ions. Salicylate and catechol were detected as metabolites of the 2-nitrobenzoate degradation using high performance liquid chromatography and gas chromatography–mass spectrometry. Enzyme activities for 2-nitrobenzoate-2-monooxygenase, salicylate hydroxylase, and catechol-1,2-dioxygenase were detected in the crude extracts of the 2-nitrobenzoate-induced cells of strain SPG. The 2-nitrobenzoate-monooxygenase activity resulted in formation of salicylate and nitrite from 2-nitrobenzoate, whereas salicylate hydroxylase catalyzed the conversion of salicylate to catechol. The ring-cleaving enzyme, catechol-1,2-dioxygenase cleaved catechol to cis,cis-muconic acid. Cells of strain SPG were able to degrade 2-nitrobenzoate in sterile as well as non-sterile soil microcosms. The results of microcosm studies showed that strain SPG degraded more than 90% of 2-nitrobenzoate within 10–12 days. This study clearly shows that Arthrobacter sp. SPG degraded 2-nitrobenzoate via a new pathway with formation of salicylate and catechol as metabolites. Arthrobacter sp. SPG may be used for bioremediation of 2-nitrobenzoate-contaminated sites due to its ability to degrade 2-nitrobenzoate in soil. PMID:26082768

  11. Sodium persulfate-assisted mechanochemical degradation of tetrabromobisphenol A: Efficacy, products and pathway.

    PubMed

    Liu, Xitao; Zhang, Xiaohui; Zhang, Kunlun; Qi, Chengdu

    2016-05-01

    In recent years, activated persulfate (PS) oxidation has been developed as a new advanced oxidation process for the degradation of organic pollutants. On the other hand, the mechanochemical method has exhibited a unique advantage in dealing with chemical wastes. The degradation of tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant (BFR), in wastes has attracted considerable attention. In this study, the efficacy of a CaO-mechanochemical (CaO-MC) treatment system assisted by the addition of PS for the degradation of TBBPA was investigated. Under the optimum reaction conditions with a mole ratio of PS:CaO = 1:4 and less than 12.5% of TBBPA by mass, the degradation and debromination of TBBPA were completed within 2 h, while the mineralization was completed within 4 h. Characterization of the milled sample by XRD revealed that CaSO4 crystallization occurred. The TG results illustrate that there was little organic matter left after 4 h of milling. Raman and FT-IR spectra exhibited the TBBPA destruction process and disappearance of the organic groups. Through analysis by LC/MS/MS, seventeen intermediates were identified. The mechanism of TBBPA degradation by the PS-assisted CaO-MC treatment system was explained from two aspects, the course of crystallization and the degradation of TBBPA by activated PS, and two parallel initiation pathways were proposed.

  12. Electron tunneling through covalent and noncovalent pathways in proteins

    NASA Technical Reports Server (NTRS)

    Beratan, David N.; Onuchic, Jose Nelson; Hopfield, J. J.

    1987-01-01

    A model is presented for electron tunneling in proteins which allows the donor-acceptor interaction to be mediated by the covalent bonds between amino acids and noncovalent contacts between amino acid chains. The important tunneling pathways are predicted to include mostly bonded groups with less favorable nonbonded interactions being important when the through bond pathway is prohibitively long. In some cases, vibrational motion of nonbonded groups along the tunneling pathway strongly influences the temperature dependence of the rate. Quantitative estimates for the sizes of these noncovalent interactions are made and their role in protein mediated electron transport is discussed.

  13. Debra, a protein mediating lysosomal degradation, is required for long-term memory in Drosophila.

    PubMed

    Kottler, Benjamin; Lampin-Saint-Amaux, Aurélie; Comas, Daniel; Preat, Thomas; Goguel, Valérie

    2011-01-01

    A central goal of neuroscience is to understand how neural circuits encode memory and guide behavior changes. Many of the molecular mechanisms underlying memory are conserved from flies to mammals, and Drosophila has been used extensively to study memory processes. To identify new genes involved in long-term memory, we screened Drosophila enhancer-trap P(Gal4) lines showing Gal4 expression in the mushroom bodies, a specialized brain structure involved in olfactory memory. This screening led to the isolation of a memory mutant that carries a P-element insertion in the debra locus. debra encodes a protein involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome. To study debra's role in memory, we achieved debra overexpression, as well as debra silencing mediated by RNA interference. Experiments conducted with a conditional driver that allowed us to specifically restrict transgene expression in the adult mushroom bodies led to a long-term memory defect. Several conclusions can be drawn from these results: i) debra levels must be precisely regulated to support normal long-term memory, ii) the role of debra in this process is physiological rather than developmental, and iii) debra is specifically required for long-term memory, as it is dispensable for earlier memory phases. Drosophila long-term memory is the only long-lasting memory phase whose formation requires de novo protein synthesis, a process underlying synaptic plasticity. It has been shown in several organisms that regulation of proteins at synapses occurs not only at translation level of but also via protein degradation, acting in remodeling synapses. Our work gives further support to a role of protein degradation in long-term memory, and suggests that the lysosome plays a role in this process.

  14. Bioactive Lysophospholipids Generated by Hepatic Lipase Degradation of Lipoproteins Lead to Complement Activation via the Classical Pathway

    PubMed Central

    Ma, Wanchao; Paik, David C.; Barile, Gaetano R.

    2014-01-01

    Purpose. We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). Methods. The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. Results. The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. Conclusions. The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of

  15. Nrdp1-mediated degradation of the gigantic IAP, BRUCE, is a novel pathway for triggering apoptosis.

    PubMed

    Qiu, Xiao-Bo; Markant, Shirley L; Yuan, Junying; Goldberg, Alfred L

    2004-02-25

    Degradation of certain inhibitor of apoptosis proteins (IAPs) appears to be critical in the initiation of apoptosis, but the factors that regulate their degradation in mammalian cells are unknown. Nrdp1/FLRF is a RING finger-containing ubiquitin ligase that catalyzes degradation of the EGF receptor family member, ErbB3. We show here that Nrdp1 associates with BRUCE/apollon, a 530 kDa membrane-associated IAP, which contains a ubiquitin-carrier protein (E2) domain. In the presence of an exogenous E2, UbcH5c, purified Nrdp1 catalyzes BRUCE ubiquitination. In vivo, overexpression of Nrdp1 promotes ubiquitination and proteasomal degradation of BRUCE. In many cell types, apoptotic stimuli induce proteasomal degradation of BRUCE (but not of XIAP or c-IAP1), and decreasing Nrdp1 levels by RNA interference reduces this loss of BRUCE. Furthermore, decreasing BRUCE content by RNA interference or overexpression of Nrdp1 promotes apoptosis. Thus, BRUCE normally inhibits apoptosis, and Nrdp1 can be important in the initiation of apoptosis by catalyzing ubiquitination and degradation of BRUCE.

  16. Nrdp1-mediated degradation of the gigantic IAP, BRUCE, is a novel pathway for triggering apoptosis

    PubMed Central

    Qiu, Xiao-Bo; Markant, Shirley L; Yuan, Junying; Goldberg, Alfred L

    2004-01-01

    Degradation of certain inhibitor of apoptosis proteins (IAPs) appears to be critical in the initiation of apoptosis, but the factors that regulate their degradation in mammalian cells are unknown. Nrdp1/FLRF is a RING finger-containing ubiquitin ligase that catalyzes degradation of the EGF receptor family member, ErbB3. We show here that Nrdp1 associates with BRUCE/apollon, a 530 kDa membrane-associated IAP, which contains a ubiquitin-carrier protein (E2) domain. In the presence of an exogenous E2, UbcH5c, purified Nrdp1 catalyzes BRUCE ubiquitination. In vivo, overexpression of Nrdp1 promotes ubiquitination and proteasomal degradation of BRUCE. In many cell types, apoptotic stimuli induce proteasomal degradation of BRUCE (but not of XIAP or c-IAP1), and decreasing Nrdp1 levels by RNA interference reduces this loss of BRUCE. Furthermore, decreasing BRUCE content by RNA interference or overexpression of Nrdp1 promotes apoptosis. Thus, BRUCE normally inhibits apoptosis, and Nrdp1 can be important in the initiation of apoptosis by catalyzing ubiquitination and degradation of BRUCE. PMID:14765125

  17. [Degradation of L-phenylalanine and of aromatic carboxylic acids by chloridazon-degrading bacteria. Combination of side chain degradation and dioxygenase pathway].

    PubMed

    Wegst, W; Lingens, F

    1981-09-01

    Strain N of Chloridazon-degrading bacteria degrades phenylalanine via cis-2,3-dihydro-2,3-dihydroxyphenylalanine,2,3-dihydroxyphenylalanine aspartate and 4-hydroxy-2-oxovalerate [Hoppe-Seyler's Z. Physiol. Chem. 360, 957--969, (1979); Biochem. J. 194, 679--684 (1981)]. cis-2,3-Dihydro-2,3-dihydroxyphenylalanine and 2,3-dihydroxyphenylalanine as well as phenylpyruvate, cis-2,3-dihydro-2,3-dihydroxyphenylpyruvate, 2,3-dihydroxyphenylpyruvate, cis-2,3-dihydro-2,3-dihydroxyphenylacetate, 2,3-dihydroxyphenylacetate and 2,3-dihydroxybenzaldehyde are detectable in the medium of strain E during growth on phenylalanine. Incubation with phenylacetate, 3-phenylpropionate or 4-phenylbutyrate leads to the accumulation of the corresponding cis-2,3-dihydro-2,3-dihydroxyphenyl derivatives. These compounds are transformed with dihydrodiol dehydrogenase to 2,3-dihydroxyphenylacetate, 3-(2,3-dihydroxyphenyl)propionate and 4-(2,3-dihydroxyphenyl)-butyrate, 3-(2,3-dihydroxyphenyl)propionate is attacked by a catechol 2,3-dioxygenase and the meta-cleavage product is again cleaved by a hydrolase yielding succinate. In a similar reaction sequence the degradation of 4-phenylbutyrate leads to the formation of glutarate. From the growth medium of strain E on phenylacetate also small amounts of 2-, 3- and 4-hydroxyphenylacetate were isolated. Resting cells were shown to metabolize 3- and 4-hydroxyphenylacetate via homogentisate and 3,4-dihydroxyphenylacetate. In the culture medium of strain K2AP benzoate could be detected. Pathways for the degradation of phenylalanine and aromatic carboxylic acids in chloridazon degrading bacteria are proposed.

  18. Coordinated Regulation of Species-Specific Hydroxycinnamic Acid Degradation and Siderophore Biosynthesis Pathways in Agrobacterium fabrum

    PubMed Central

    Baude, Jessica; Vial, Ludovic; Villard, Camille; Campillo, Tony; Lavire, Céline; Nesme, Xavier

    2016-01-01

    ABSTRACT The rhizosphere-inhabiting species Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to degrade hydroxycinnamic acids (HCAs), especially ferulic acid and p-coumaric acid, via the novel A. fabrum HCA degradation pathway. Gene expression profiles of A. fabrum strain C58 were investigated in the presence of HCAs, using a C58 whole-genome oligoarray. Both ferulic acid and p-coumaric acid caused variations in the expression of more than 10% of the C58 genes. Genes of the A. fabrum HCA degradation pathway, together with the genes involved in iron acquisition, were among the most highly induced in the presence of HCAs. Two operons coding for the biosynthesis of a particular siderophore, as well as genes of the A. fabrum HCA degradation pathway, have been described as being specific to the species. We demonstrate here their coordinated expression, emphasizing the interdependence between the iron concentration in the growth medium and the rate at which ferulic acid is degraded by cells. The coordinated expression of these functions may be advantageous in HCA-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. The present results confirm that there is cooperation between the A. fabrum-specific genes, defining a particular ecological niche. IMPORTANCE We previously identified seven genomic regions in Agrobacterium fabrum that were specifically present in all of the members of this species only. Here we demonstrated that two of these regions, encoding the hydroxycinnamic acid degradation pathway and the iron acquisition pathway, were regulated in a coordinated manner. The coexpression of these functions may be advantageous in hydroxycinnamic acid-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. These data support the view that bacterial genomic species

  19. Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots

    PubMed Central

    Zhang, Hongtao; Gannon, Lucy; Powers, Stephen J; Lilley, Kathryn S; Theodoulou, Frederica L

    2015-01-01

    According to the Arg/N-end rule pathway, proteins with basic N-termini are targeted for degradation by the Arabidopsis thaliana E3 ligase, PROTEOLYSIS6 (PRT6). Proteins can also become PRT6 substrates following post-translational arginylation by arginyltransferases ATE1 and 2. Here, we undertook a quantitative proteomics study of Arg/N-end rule mutants, ate1/2 and prt6, to investigate the impact of this pathway on the root proteome. Tandem mass tag labelling identified a small number of proteins with increased abundance in the mutants, some of which represent downstream targets of transcription factors known to be N-end rule substrates. Isolation of N-terminal peptides using terminal amine isotope labelling of samples (TAILS) combined with triple dimethyl labelling identified 1465 unique N-termini. Stabilising residues were over-represented among the free neo-N-termini, but destabilising residues were not markedly enriched in N-end rule mutants. The majority of free neo-N-termini were revealed following cleavage of organellar targeting signals, thus compartmentation may account in part for the presence of destabilising residues in the wild-type N-terminome. Our data suggest that PRT6 does not have a marked impact on the global proteome of Arabidopsis roots and is likely involved in the controlled degradation of relatively few regulatory proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD001719 (http://proteomecentral.proteomexchange.org/dataset/PXD001719). PMID:25728785

  20. Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

    PubMed Central

    Son, Sung Min; Cha, Moon-Yong; Choi, Heesun; Kang, Seokjo; Choi, Hyunjung; Lee, Myung-Shik; Park, Sun Ah; Mook-Jung, Inhee

    2016-01-01

    ABSTRACT The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology. PMID:26963025

  1. Metabolic pathway of 3,6-anhydro-D-galactose in carrageenan-degrading microorganisms.

    PubMed

    Lee, Sun Bok; Kim, Jeong Ah; Lim, Hyun Seung

    2016-05-01

    Complete hydrolysis of κ-carrageenan produces two sugars, D-galactose and 3,6-anhydro-D-galactose (D-AnG). At present, however, we do not know how carrageenan-degrading microorganisms metabolize D-AnG. In this study, we investigated the metabolic pathway of D-AnG degradation by comparative genomic analysis of Cellulophaga lytica LIM-21, Pseudoalteromonas atlantica T6c, and Epulopiscium sp. N.t. morphotype B, which represent the classes Flavobacteria, Gammaproteobacteria, and Clostridia, respectively. In this bioinformatic analysis, we found candidate common genes that were believed to be involved in D-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the D-AnG cluster. In all three microorganisms, D-AnG metabolizing genes were clustered and organized in operon-like arrangements, which we named as the dan operon (3,6-d-anhydro-galactose). Combining bioinformatic analysis and experimental data, we showed that D-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via four enzyme-catalyzed reactions in the following route: 3,6-anhydro-D-galactose → 3,6-anhydro-D-galactonate → 2-keto-3-deoxy-D-galactonate (D-KDGal) → 2-keto-3-deoxy-6-phospho-D-galactonate → pyruvate + D-glyceraldehyde-3-phosphate. The pathway of D-AnG degradation is composed of two parts: transformation of D-AnG to D-KDGal using two D-AnG specific enzymes and breakdown of D-KDGal to two glycolysis intermediates using two DeLey-Doudoroff pathway enzymes. To our knowledge, this is the first report on the metabolic pathway of D-AnG degradation.

  2. Epidermal growth factor receptor degradation: an alternative view of oncogenic pathways.

    PubMed

    Kirisits, Andreas; Pils, Dietmar; Krainer, Michael

    2007-01-01

    Positive regulation of epidermal growth factor receptor signalling is related to many human malignancies. Besides overexpression and gain of function mutations, the escape from negative regulation through an increase in epidermal growth factor receptor stability has evolved as yet another key factor contributing to enhanced receptor activity. Intensive research over the past years has provided considerable evidence concerning the molecular mechanisms which provide epidermal growth factor receptor degradation. c-Cbl mediated ubiquitination, endocytosis via clathrin-coated pits, endosomal sorting and lysosomal degradation have become well-investigated cornerstones. Recent findings on the interdependency of the endosomal sorting complexes required for transport in multivesicular body sorting, stress the topicality of receptor tyrosine kinase downregulation. Here, we review the degradation pathway of the epidermal growth factor receptor, following the receptor from ligand binding to the lysosome and illustrating different modes of oncogenic deregulation.

  3. Anaerobic degradation pathway of linear Alkylbenzene sulfonates (LAS) in sulfate-reducing marine sediments.

    PubMed

    Lara-Martín, Pablo A; Gómez-Parra, Abelardo; Sanz, José Luis; González-Mazo, Eduardo

    2010-03-01

    Linear alkylbenzene sulfonates (LAS) are among the principal synthetic surfactants used worldwide. Their presence in the environment has been reported in a significant number of studies, and it has been generally assumed that LAS are not biotransformed in the absence of oxygen. However, laboratory experiments performed by our group using anoxic marine sediments have reported LAS degradation percentages that can reach up to 79% in 165 days. Here, we show for the first time the initial reaction metabolites (generated via fumarate addition to the LAS molecules), their biotransformation into sulfophenyl carboxylic acids (SPC), and the progressive degradation of these by successive beta-oxidation reactions. Advanced mass spectrometry has been used to carry out the identification of these compounds. This is the first time that an anaerobic degradation pathway for LAS is described, and these results represent a significant advance in understanding the final fate of these and other similar compounds in anoxic environments.

  4. Photocatalytic degradation of gaseous 1-propanol using an annular reactor: kinetic modelling and pathways.

    PubMed

    Vincent, G; Marquaire, P M; Zahraa, O

    2009-01-30

    Photocatalytic oxidation of airborne contaminants appears to be a promising process for remediation of air polluted by Volatile Organic Compounds (VOCs). In the present work, the photocatalytic oxidation of gaseous 1-propanol has been investigated by using an annular photoreactor. The annular photocatalytic reactor was modelled by a cascade of heightened elementary continuously stirred tank reactors. The influence of several kinetic parameters such as pollutant concentration, incident light irradiance, contact time and humidity content has been studied. The photocatalytic degradation by-products of 1-propanol has been identified in the gas-phase by GC/MS. Propionaldehyde and acetaldehyde were found to be the main gaseous intermediates. Propionaldehyde and acetaldehyde have been taken into account in a "two-site model" to evaluate the possible competition of adsorption between 1-propanol and its by-products of degradation. A mechanistic pathway is then proposed for the photocatalytic degradation of 1-propanol.

  5. Eosinophil granule cationic proteins regulate the classical pathway of complement.

    PubMed Central

    Weiler, J M; Edens, R E; Bell, C S; Gleich, G J

    1995-01-01

    Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by

  6. Novel degradation pathway and kinetic analysis for buprofezin removal by newly isolated Bacillus sp.

    PubMed

    Wang, Guangli; Xu, Dayong; Xiong, Minghua; Zhang, Hui; Li, Feng; Liu, Yuan

    2016-09-15

    Given the intensive and widespread application of the pesticide, buprofezin, its environmental residues potentially pose a problem; yet little is known about buprofezin's kinetic and metabolic behaviors. In this study, a novel gram-positive strain, designated BF-5, isolated from aerobic activated sludge, was found to be capable of metabolizing buprofezin as its sole energy, carbon, and nitrogen source. Based on its physiological and biochemical characteristics, other aspects of its phenotype, and a phylogenetic analysis, strain BF-5 was identified as Bacillus sp. This study investigated the effect of culture conditions on bacterial growth and substrate degradation, such as pH, temperature, initial concentration, different nitrogen source, and additional nitrogen sources as co-substrates. The degradation rate parameters, qmax, Ks, Ki and Sm were determined to be 0.6918 h(-1), 105.4 mg L(-1), 210.5 mg L(-1), and 148.95 mg L(-1) respectively. The capture of unpublished potential metabolites by gas chromatography-mass spectrometry (GC-MS) analysis has led to the proposal of a novel degradation pathway. Taken together, our results clarify buprofezin's biodegradation pathway(s) and highlight the promising potential of strain BF-5 in bioremediation of buprofezin-contaminated environments.

  7. Effects of chlorobenzoate transformation on the Pseudomonas testosteroni biphenyl and chlorobiphenyl degradation pathway

    SciTech Connect

    Sondossi, M.; Sylvestre, M.; Ahmad, D. )

    1992-02-01

    Bacterial conversion of biphenyl (BP) and chlorobiphenyls (CBPs) to benzoates and chlorobenzoates (CBAs) proceeds by introduction of molecular oxygen at the 2,3 position, followed by a 1,2-meta cleavage of the molecule. Complete mineralization of CBPs requires the presence of two sets of genes, one for the transformation of CBPs into CBAs and a second for the degradation of CBAs. It has been shown previously that removal of the CBAs produced from the degradation of CBPs is essential for efficient degradation of CBPs. In this study the authors confirmed that CBAs inhibit BP and CBP transformation in Pseudomonas testosteroni B-356. Among the three monochlorobenzoates tested, 3-chlorobenzoate was the most effective inhibitor. Furthermore, they found that in strain B-356, CBA transformation is controlled by BP-induced oxygenases that are not present in benzoate-grown cells. They found that this BP-linked CBA transformation pathway transformed CBAs produced from CBPs into several metabolites, including chlorocatechols and corresponding muconic semialdehydes. These metabolites inhibited the 2,3-dihydroxybiphenyl 1,2-dioxygenase, while CBAs by themselves had no effect on this enzyme. Therefore, on the basis of this and other observations, it appears that when CBAs produced from CBPs accumulate in the growth medium, they are converted into unproductive metabolites that reduce the flux of the BP and CBP degradation pathway.

  8. Degradation of methiocarb by monochloramine in water treatment: kinetics and pathways.

    PubMed

    Qiang, Zhimin; Tian, Fang; Liu, Wenjun; Liu, Chao

    2014-03-01

    The micropollution of drinking water sources with pesticides has become a global concern. This work investigated the degradation of methiocarb (MC), a most commonly-used carbamate pesticide, by monochloramine (NH2Cl) under simulated water treatment conditions. Results indicate that the reaction was of first-order in MC and varied orders in NH2Cl depending on water pH. The observed rate constant of MC degradation decreased quickly with either a decrease in the molar ratio of chlorine to ammonia (Cl2:N) or an increase in water pH. The apparent activation energy of the reaction was determined to be 34 kJ mol(-1). The MC degradation pathways also exhibited a strong pH dependence: at pH 6.5, MC was first oxidized by NH2Cl to methiocarb sulfoxide (MCX), and then hydrolyzed to methiocarb sulfoxide phenol (MCXP); while at pH 8.5, MCX, MCXP and methiocarb sulfone phenol (MCNP) were formed successively through either oxidation or hydrolysis reactions. Based on the identified byproducts and their concentrations evolution, the proposed pathways of MC degradation in the presence of NH2Cl were further validated through kinetic model simulations.

  9. Entner–Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1

    PubMed Central

    Felux, Ann-Katrin; Spiteller, Dieter; Klebensberger, Janosch; Schleheck, David

    2015-01-01

    Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a “sulfoglycolytic” pathway, in addition to the classical glycolytic (Embden–Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner–Doudoroff pathway for glucose-6-phosphate: It involves an NAD+-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)+-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium. PMID:26195800

  10. Entner-Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1.

    PubMed

    Felux, Ann-Katrin; Spiteller, Dieter; Klebensberger, Janosch; Schleheck, David

    2015-08-04

    Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a "sulfoglycolytic" pathway, in addition to the classical glycolytic (Embden-Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner-Doudoroff pathway for glucose-6-phosphate: It involves an NAD(+)-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)(+)-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.

  11. Reading normal and degraded words: contribution of the dorsal and ventral visual pathways.

    PubMed

    Cohen, Laurent; Dehaene, Stanislas; Vinckier, Fabien; Jobert, Antoinette; Montavont, Alexandra

    2008-03-01

    Fast, parallel word recognition, in expert readers, relies on sectors of the left ventral occipito-temporal pathway collectively known as the visual word form area. This expertise is thought to arise from perceptual learning mechanisms that extract informative features from the input strings. The perceptual expertise hypothesis leads to two predictions: (1) parallel word recognition, based on the ventral visual system, should be limited to words displayed in a familiar format (foveal horizontal words with normally spaced letters); (2) words displayed in formats outside this field of expertise should be read serially, under supervision of dorsal parietal attention systems. We presented adult readers with words that were progressively degraded in three different ways (word rotation, letter spacing, and displacement to the visual periphery). Behaviorally, we identified degradation thresholds above which reading difficulty increased non-linearly, with the concomitant emergence of a word length effect on reading latencies reflecting serial reading strategies. fMRI activations were correlated with reading difficulty in bilateral occipito-temporal and parietal regions, reflecting the strategies required to identify degraded words. A core region of the intraparietal cortex was engaged in all modes of degradation. Furthermore, in the ventral pathway, word degradation led to an amplification of activation in the posterior visual word form area, at a level thought to encode single letters. We also found an effect of word length restricted to highly degraded words in bilateral occipitoparietal regions. Those results clarify when and how the ventral parallel visual word form system needs to be supplemented by the deployment of dorsal serial reading strategies.

  12. Evidence of oleuropein degradation by olive leaf protein extract.

    PubMed

    De Leonardis, Antonella; Macciola, Vincenzo; Cuomo, Francesca; Lopez, Francesco

    2015-05-15

    The enzymatic activity of raw protein olive leaf extract has been investigated in vivo, on olive leaf homogenate and, in vitro with pure oleuropein and other phenolic substrates. At least two types of enzymes were found to be involved in the degradation of endogenous oleuropein in olive leaves. As for the in vitro experiments, the presence of active polyphenoloxidase and β-glucosidase was determined by HPLC and UV-Visible spectroscopy. Interestingly, both the enzymatic activities were found to change during the storage of olive leaves. Specifically, the protein extracts obtained from fresh leaves showed the presence of both the enzymatic activities, because oleuropein depletion occurred simultaneously with the formation of the oleuropein aglycon, 3,4-DHPEA-EA. In comparison leaves subjected to the drying process showed a polyphenoloxidase activity leading exclusively to the formation of oxidation products responsible for the typical brown coloration of the reaction solution.

  13. Porcine arterivirus activates the NF-{kappa}B pathway through I{kappa}B degradation

    SciTech Connect

    Lee, Sang-Myeong; Kleiboeker, Steven B. . E-mail: KleiboekerS@Missouri.edu

    2005-11-10

    Nuclear factor-kappaB (NF-{kappa}B) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-{kappa}B in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-{kappa}B activation was characterized by translocation of NF-{kappa}B from the cytoplasm to the nucleus, increased DNA binding activity, and NF-{kappa}B-regulated gene expression. NF-{kappa}B activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-{kappa}B activation. Degradation of I{kappa}B protein was detected late in PRRSV infection, and overexpression of the dominant negative form of I{kappa}B{alpha} (I{kappa}B{alpha}DN) significantly suppressed NF-{kappa}B activation induced by PRRSV. However, I{kappa}B{alpha}DN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-{kappa}B DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-{kappa}B was activated by PRRSV infection. Moreover, NF-{kappa}B-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-{kappa}B activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.

  14. Synthetic metabolism: engineering biology at the protein and pathway scales.

    PubMed

    Martin, Collin H; Nielsen, David R; Solomon, Kevin V; Prather, Kristala L Jones

    2009-03-27

    Biocatalysis has become a powerful tool for the synthesis of high-value compounds, particularly so in the case of highly functionalized and/or stereoactive products. Nature has supplied thousands of enzymes and assembled them into numerous metabolic pathways. Although these native pathways can be use to produce natural bioproducts, there are many valuable and useful compounds that have no known natural biochemical route. Consequently, there is a need for both unnatural metabolic pathways and novel enzymatic activities upon which these pathways can be built. Here, we review the theoretical and experimental strategies for engineering synthetic metabolic pathways at the protein and pathway scales, and highlight the challenges that this subfield of synthetic biology currently faces.

  15. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    PubMed Central

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  16. Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation.

    PubMed

    Gao, Caiji; Zhuang, Xiaohong; Cui, Yong; Fu, Xi; He, Yilin; Zhao, Qiong; Zeng, Yonglun; Shen, Jinbo; Luo, Ming; Jiang, Liwen

    2015-02-10

    Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 domain-containing protein2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.

  17. Role of the crc gene in catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathway.

    PubMed

    Yuste, L; Rojo, F

    2001-11-01

    Expression of the alkane degradation pathway encoded in the OCT plasmid of Pseudomonas putida GPo1 is induced in the presence of alkanes by the AlkS regulator, and it is down-regulated by catabolic repression. The catabolic repression effect reduces the expression of the two AlkS-activated promoters of the pathway, named PalkB and PalkS2. The P. putida Crc protein participates in catabolic repression of some metabolic pathways for sugars and nitrogenated compounds. Here, we show that Crc has an important role in the catabolic repression exerted on the P. putida GPo1 alkane degradation pathway when cells grow exponentially in a rich medium. Interestingly, Crc plays little or no role on the catabolic repression exerted by some organic acids in a defined medium, which shows that these two types of catabolic repression can be genetically distinguished. Disruption of the crc gene led to a six- to sevenfold increase in the levels of the mRNAs arising from the AlkS-activated PalkB and PalkS2 promoters in cells growing exponentially in rich medium. This was not due to an increase in the half-lives of these mRNAs. Since AlkS activates the expression of its own gene and seems to be present in limiting amounts, the higher mRNA levels observed in the absence of Crc could arise from an increase in either transcription initiation or in the translation efficiency of the alkS mRNA. Both alternatives would lead to increased AlkS levels and hence to elevated expression of PalkB and PalkS2. High expression of alkS from a heterologous promoter eliminated catabolic repression. Our results indicate that catabolic repression in rich medium is directed to down-regulate the levels of the AlkS activator. Crc would thus modulate, directly or indirectly, the levels of AlkS.

  18. Dissection of the Dislocation Pathway for Type I Membrane Proteins with a New Small Molecule Inhibitor, Eeyarestatin

    PubMed Central

    Fiebiger, Edda; Hirsch, Christian; Vyas, Jatin M.; Gordon, Eva; Ploegh, Hidde L.; Tortorella, Domenico

    2004-01-01

    The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor α. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation. PMID:14767067

  19. Degradation of protein disulphide bonds in dilute alkali.

    PubMed Central

    Florence, T M

    1980-01-01

    The degradation of S--S bonds in 0.2 M-NaOH at 25 degrees C was studied for a series of proteins and simple aliphatic disulphide compounds, by using cathodic stripping voltammetry, ion-selective-electrode potentiometry, spectrophotometry and ultrafiltration. The disulphide bonds that dissociated in 0.2 M-NaOH were usually those that are solvent accessible and that can be reduced by mild chemical reductants. Some unexpected differences were found between similar proteins, both in the number of S--S bonds dissociated and in their rates of decomposition. Chymotrypsin has one S--S bond attacked, whereas chymotrypsinogen and trypsinogen have two. Ribonuclease A has two S--S bonds dissociated, but ribonuclease S and S-protein have three. Denaturation in 6 M-guanidine hydrochloride before alkaline digestion caused the loss of an additional S--S bond in ribonuclease A and insulin, and increased the rate of dissociation of the S--S bonds of some other proteins. The initial product of S--S bond dissociation in dilute alkali is believed to be a persulphide intermediate formed by a beta-elimination reaction. This intermediate is in mobile equilibrium with bisulphide ion, HS-, and decomposes at a mercury electrode or in acid solution to yield a stoichiometric amount of sulphide. Rate constants and equilibrium constants were measured for the equilibria between HS- and the intermediates involved in the alkaline dissociation of several proteins. Elemental sulphur was not detected in any of the protein digests. It is suggested that formation of HS- from a persulphide intermediate involves a hydrolysis reaction to yield a sulphenic acid derivative. The small polypeptides glutathione and oxytocin gave only a low yield of persulphide, and their alkaline decomposition must proceed by a mechanism different from that of the proteins. PMID:7213343

  20. Comparative genomic analysis of nine Sphingobium strains: Insights into their evolution and hexachlorocyclohexane (HCH) degradation pathways

    DOE PAGES

    Verma, Helianthous; Kumar, Roshan; Oldach, Phoebe; ...

    2014-11-23

    Background: Sphingobium spp. are efficient degraders of a wide range of chlorinated and aromatic hydrocarbons. In particular, strains which harbour the lin pathway genes mediating the degradation of hexachlorocyclohexane (HCH) isomers are of interest due to the widespread persistence of this contaminant. Here, we examined the evolution and diversification of the lin pathway under the selective pressure of HCH, by comparing the draft genomes of six newly-sequenced Sphingobium spp. (strains LL03, DS20, IP26, HDIPO4, P25 and RL3) isolated from HCH dumpsites, with three existing genomes (S. indicum B90A, S. japonicum UT26S and Sphingobium sp. SYK6). Results: Efficient HCH degraders phylogeneticallymore » clustered in a closely related group comprising of UT26S, B90A, HDIPO4 and IP26, where HDIPO4 and IP26 were classified as subspecies with ANI value >98%. Less than 10% of the total gene content was shared among all nine strains, but among the eight HCH-associated strains, that is all except SYK6, the shared gene content jumped to nearly 25%. Genes associated with nitrogen stress response and two-component systems were found to be enriched. The strains also housed many xenobiotic degradation pathways other than HCH, despite the absence of these xenobiotics from isolation sources. In addition, these strains, although non-motile, but posses flagellar assembly genes. While strains HDIPO4 and IP26 contained the complete set of lin genes, DS20 was entirely devoid of lin genes (except linKLMN) whereas, LL03, P25 and RL3 were identified as lin deficient strains, as they housed incomplete lin pathways. Further, in HDIPO4, linA was found as a hybrid of two natural variants i.e., linA1 and linA2 known for their different enantioselectivity. In conclusion, the bacteria isolated from HCH dumpsites provide a natural testing ground to study variations in the lin system and their effects on degradation efficacy. Further, the diversity in the lin gene sequences and copy number, their

  1. Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

    PubMed Central

    van der Werf, Mariët J.; Swarts, Henk J.; de Bont, Jan A. M.

    1999-01-01

    Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In

  2. Styrene lower catabolic pathway in Pseudomonas fluorescens ST: identification and characterization of genes for phenylacetic acid degradation.

    PubMed

    Di Gennaro, Patrizia; Ferrara, Silvia; Ronco, Ilaria; Galli, Enrica; Sello, Guido; Papacchini, Maddalena; Bestetti, Giuseppina

    2007-08-01

    Pseudomonas fluorescens ST is a styrene degrading microorganism that, by the sequential oxidation of the vinyl side chain, converts styrene to phenylacetic acid. The cluster of styrene upper pathway catabolic genes (sty genes) has been previously localized on a chromosomal region. This report describes the isolation, sequencing and analysis of a new chromosomal fragment deriving from the ST strain genomic bank that contains the styrene lower degradative pathway genes (paa genes), involved in the metabolism of phenylacetic acid. Analysis of the paa gene cluster led to the description of 14 putative genes: a gene encoding a phenylacetyl-CoA ligase (paaF), the enzyme required for the activation of phenylacetic acid; five ORFs encoding the subunits of a ring hydroxylation multienzymatic system (paaGHIJK); the gene paaW encoding a membrane protein of unknown function; five genes for a beta-oxidation-like system (paaABCDE), involved in the steps following the aromatic ring cleavage; a gene encoding a putative permease (paaL) and a gene (paaN) probably involved in the aromatic ring cleavage. The function of some of the isolated genes has been proved by means of biotransformation experiments.

  3. Mutations in NGLY1 Cause an Inherited Disorder of the Endoplasmic Reticulum-Associated Degradation (ERAD) Pathway

    PubMed Central

    Enns, Gregory M.; Shashi, Vandana; Bainbridge, Matthew; Gambello, Michael J.; Zahir, Farah R.; Bast, Thomas; Crimian, Rebecca; Schoch, Kelly; Platt, Julia; Cox, Rachel; Bernstein, Jonathan; Scavina, Mena; Walter, Rhonda S.; Bibb, Audrey; Jones, Melanie; Hegde, Madhuri; Graham, Brett H.; Need, Anna C.; Oviedo, Angelica; Schaaf, Christian P.; Boyle, Sean; Butte, Atul J.; Chen, Rong; Clark, Michael J.; Haraksingh, Rajini; Cowan, Tina M.; He, Ping; Langlois, Sylvie; Zoghbi, Huda Y.; Snyder, Michael; Gibbs, Richard; Freeze, Hudson H.; Goldstein, David B.

    2014-01-01

    Purpose The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for the translocation of misfolded proteins across the ER membrane into the cytosol for subsequent degradation by the proteasome. In order to understand the spectrum of clinical and molecular findings in a complex neurological syndrome, we studied a series of eight patients with inherited deficiency of N-glycanase 1 (NGLY1), a novel disorder of cytosolic ERAD dysfunction. Methods Whole-genome, whole-exome or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data. Results All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypo- or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele. Conclusions NGLY1 deficiency is a novel autosomal recessive disorder of the ERAD pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a more broad range of mutations are detected. PMID:24651605

  4. Non-repair Pathways for Minimizing Protein Isoaspartyl Damage in the Yeast Saccharomyces cerevisiae*

    PubMed Central

    Patananan, Alexander N.; Capri, Joseph; Whitelegge, Julian P.; Clarke, Steven G.

    2014-01-01

    The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50–300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis. PMID:24764295

  5. Non-repair pathways for minimizing protein isoaspartyl damage in the yeast Saccharomyces cerevisiae.

    PubMed

    Patananan, Alexander N; Capri, Joseph; Whitelegge, Julian P; Clarke, Steven G

    2014-06-13

    The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50-300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.

  6. From ether to acid: A plausible degradation pathway of glycerol dialkyl glycerol tetraethers

    NASA Astrophysics Data System (ADS)

    Liu, Xiao-Lei; Birgel, Daniel; Elling, Felix J.; Sutton, Paul A.; Lipp, Julius S.; Zhu, Rong; Zhang, Chuanlun; Könneke, Martin; Peckmann, Jörn; Rowland, Steven J.; Summons, Roger E.; Hinrichs, Kai-Uwe

    2016-06-01

    Glycerol dialkyl glycerol tetraethers (GDGTs) are ubiquitous microbial lipids with extensive demonstrated and potential roles as paleoenvironmental proxies. Despite the great attention they receive, comparatively little is known regarding their diagenetic fate. Putative degradation products of GDGTs, identified as hydroxyl and carboxyl derivatives, were detected in lipid extracts of marine sediment, seep carbonate, hot spring sediment and cells of the marine thaumarchaeon Nitrosopumilus maritimus. The distribution of GDGT degradation products in environmental samples suggests that both biotic and abiotic processes act as sinks for GDGTs. More than a hundred newly recognized degradation products afford a view of the stepwise degradation of GDGT via (1) ether bond hydrolysis yielding hydroxyl isoprenoids, namely, GDGTol (glycerol dialkyl glycerol triether alcohol), GMGD (glycerol monobiphytanyl glycerol diether), GDD (glycerol dibiphytanol diether), GMM (glycerol monobiphytanol monoether) and bpdiol (biphytanic diol); (2) oxidation of isoprenoidal alcohols into corresponding carboxyl derivatives and (3) chain shortening to yield C39 and smaller isoprenoids. This plausible GDGT degradation pathway from glycerol ethers to isoprenoidal fatty acids provides the link to commonly detected head-to-head linked long chain isoprenoidal hydrocarbons in petroleum and sediment samples. The problematic C80 to C82 tetraacids that cause naphthenate deposits in some oil production facilities can be generated from H-shaped glycerol monoalkyl glycerol tetraethers (GMGTs) following the same process, as indicated by the distribution of related derivatives in hydrothermally influenced sediments.

  7. Oxidative degradation of N-Nitrosopyrrolidine by the ozone/UV process: Kinetics and pathways.

    PubMed

    Chen, Zhi; Fang, Jingyun; Fan, Chihhao; Shang, Chii

    2016-05-01

    N-Nitrosopyrrolidine (NPYR) is an emerging contaminant in drinking water and wastewater. The degradation kinetics and mechanisms of NPYR degradation by the O3/UV process were investigated and compared with those of UV direct photolysis and ozonation. A synergistic effect of ozone and UV was observed in the degradation of NPYR due to the accelerated production of OH• by ozone photolysis. This effect was more pronounced at higher ozone dosages. The second-order rate constants of NPYR reacting with OH• and ozone was determined to be 1.38 (± 0.05) × 10(9) M(-1) s(-1) and 0.31 (± 0.02) M(-1) s(-1), respectively. The quantum yield by direct UV photolysis was 0.3 (± 0.01). An empirical model using Rct (the ratio of the exposure of OH• to that of ozone) was established for NPYR degradation in treated drinking water and showed that the contributions of direct UV photolysis and OH• oxidation on NPYR degradation were both significant. As the reaction proceeded, the contribution by OH• became less important due to the exhausting of ozone. Nitrate was the major product in the O3/UV process by two possible pathways. One is through the cleavage of nitroso group to form NO• followed by hydrolysis, and the other is the oxidation of the intermediates of amines by ozonation.

  8. Carbon Nanotube Degradation in Macrophages: Live Nanoscale Monitoring and Understanding of Biological Pathway.

    PubMed

    Elgrabli, Dan; Dachraoui, Walid; Ménard-Moyon, Cécilia; Liu, Xiao Jie; Bégin, Dominique; Bégin-Colin, Sylvie; Bianco, Alberto; Gazeau, Florence; Alloyeau, Damien

    2015-10-27

    Despite numerous applications, the cellular-clearance mechanism of multiwalled carbon nanotubes (MWCNTs) has not been clearly established yet. Previous in vitro studies showed the ability of oxidative enzymes to induce nanotube degradation. Interestingly, these enzymes have the common capacity to produce reactive oxygen species (ROS). Here, we combined material and life science approaches for revealing an intracellular way taken by macrophages to degrade carbon nanotubes. We report the in situ monitoring of ROS-mediated MWCNT degradation by liquid-cell transmission electron microscopy. Two degradation mechanisms induced by hydroxyl radicals were extracted from these unseen dynamic nanoscale investigations: a non-site-specific thinning process of the walls and a site-specific transversal drilling process on pre-existing defects of nanotubes. Remarkably, similar ROS-induced structural injuries were observed on MWCNTs after aging into macrophages from 1 to 7 days. Beside unraveling oxidative transformations of MWCNT structure, we elucidated an important, albeit not exclusive, biological pathway for MWCNT degradation in macrophages, involving NOX2 complex activation, superoxide production, and hydroxyl radical attack, which highlights the critical role of oxidative stress in cellular processing of MWCNTs.

  9. Regulation of global protein translation and protein degradation in aerobic dormancy.

    PubMed

    Ramnanan, Christopher J; Allan, Marcus E; Groom, Amy G; Storey, Kenneth B

    2009-03-01

    We hypothesized that protein turnover would be substantially suppressed during estivation in the land snail, Otala lactea, as part of a wholesale move to conserve ATP in the hypometabolic state, and that decreased rates of protein synthesis and degradation would be mediated by altering the phosphorylation state of key proteins. Rates of protein translation, measured in vitro, decreased by approximately 80% in extracts of foot muscle and hepatopancreas after 2 days of estivation, and this reduction was associated with strong increases in the phosphorylation of ribosomal factors, eIF2 alpha and eEF2, as well as decreased phosphorylation of 4E-BP1. Reductions in levels of markers of ribosomal biogenesis and a tissue-specific reduction in the phosphorylation state of eIF4E and eIF4GI were also evident after 14 days of estivation. Activity of the 20S proteasome decreased by 60-80% after 2 days of estivation and this decrease was mediated by protein kinase G in vitro, whereas protein phosphatase 2A activated the proteasome. Levels of protein carbonyls did not change in snail tissues during estivation whereas the expression heat shock proteins increased, suggesting that protein resistance to damage is enhanced in estivation. In conclusion, protein synthesis and degradation rates were coordinately suppressed during estivation in O. lactea and this is associated with the phosphorylation of ribosomal initiation and elongation factors and the 20S proteasome.

  10. L-Arabinose degradation pathway in the haloarchaeon Haloferax volcanii involves a novel type of L-arabinose dehydrogenase.

    PubMed

    Johnsen, Ulrike; Sutter, Jan-Moritz; Zaiß, Henning; Schönheit, Peter

    2013-11-01

    The pathway of L-arabinose degradation was studied in the haloarchaeon Haloferax volcanii. It is shown that L-arabinose is oxidatively degraded to α-ketoglutarate. During growth on L-arabinose, L-arabinose dehydrogenase (L-AraDH) was induced. The enzyme was purified as a 130 kDa homotetrameric protein catalyzing the oxidation of L-arabinose with both NADP(+) and NAD(+). The gene encoding L-AraDH was identified as HVO_B0032 and recombinant L-AraDH showed similar properties as the native enzyme. The L-AraDH deletion mutant did not grow on L-arabinose, but grew unaffected on glucose and D-xylose, indicating a specific involvement in L-arabinose degradation. Phylogenetic analyses attribute the first archaeal L-AraDH to the extended short-chain dehydrogenase/reductase (SDRe) family, where it is part of a novel cluster and thus differs from known archaeal and bacterial pentose dehydrogenases. Further, cell extracts of H. volcanii catalyzed the NADP(+)-dependent conversion of L-arabinoate to α-ketoglutarate. The genes involved in that conversion were identified by analyses of transcripts and deletion mutants as HVO_B0038A, HVO_B0027 and HVO_B0039 recently reported to be involved in D-xylonate conversion to α-ketoglutarate in H. volcanii (Johnsen et al. 2009).

  11. Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However,...

  12. Anoxic Androgen Degradation by the Denitrifying Bacterium Sterolibacterium denitrificans via the 2,3-seco Pathway

    PubMed Central

    Wang, Po-Hsiang; Yu, Chang-Ping; Lee, Tzong-Huei; Lin, Ching-Wen; Ismail, Wael; Wey, Shiaw-Pyng; Kuo, An-Ti

    2014-01-01

    The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C19 androgens. By using a 13C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the identification of a C17 intermediate, we propose that the A-ring cleavage may be followed by the removal of a C2 side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (≤500 μM) did not exhibit androgenic activity, whereas the sterane-containing intermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteobacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, and both of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acid can be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C19 androgens. PMID:24657867

  13. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    PubMed Central

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  14. The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway.

    PubMed

    Suzuki, T; Park, H; Till, E A; Lennarz, W J

    2001-10-12

    Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.

  15. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion.

    PubMed

    Ling Lin Fu; Zi Rong Xu; Wei Fen Li; Jiang Bing Shuai; Ping Lu; Chun Xia Hu

    2007-01-01

    The absence of an outer membrane in Bacillus subtilis can simplify the protein secretion pathways and allow the organism to secrete high levels of extracellular proteins. Of the three known secretory routes, Sec-SRP pathway can direct the majority of secretory proteins into the growth medium. Alternatively, a small number of exoproteins with specific functions are secreted via Tat pathway or ABC transporters in B. subtilis. The discriminating function of precursor proteins among these pathways is largely attributed to the distinct structure of their cleavable signal peptides. Individual secretion machinery components with their special functions are involved in the total flow of proteins from the cytoplasm to the medium. Notably, multiple regulators with signal transduction functions can affect expression of secretion machinery as well as their post-transcriptional actions for protein secretion, resulting in the complicated networks in B. subtilis. Ultimately, according to the available knowledge of secretion machinery, several approaches aimed at optimizing protein secretion are discussed.

  16. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  17. Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

    PubMed

    Chen, Baolin; Wu, Qiang; Xiong, Zhaojun; Ma, Yuedong; Yu, Sha; Chen, Dandan; Huang, Shengwen; Dong, Yugang

    2016-09-01

    Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway.

  18. Lentiviral Effector Pathways of TRIM Proteins.

    PubMed

    Turrini, Filippo; Di Pietro, Andrea; Vicenzi, Elisa

    2014-04-01

    The human tripartite motif (TRIM) family, composed of more than 77 members, encompasses an emerging group of innate antiviral factors. Most TRIM proteins are characterized by being E3 ubiquitin ligases, but also engage in specific interactions with a variety of cellular and viral partners. They are involved in many cellular processes, including cell differentiation, transcriptional regulation, cytoskeleton remodeling, intracellular trafficking, membrane repair, and oncogenesis. In regard to antiviral immunity, they restrict both retroviruses and lentiviruses as well as other DNA and RNA viruses. This review will focus on the TRIM members endowed with anti-retroviral and anti-lentiviral activities and, in particular, human immunodeficiency virus.

  19. Stress responses during ageing: molecular pathways regulating protein homeostasis.

    PubMed

    Kyriakakis, Emmanouil; Princz, Andrea; Tavernarakis, Nektarios

    2015-01-01

    The ageing process is characterized by deterioration of physiological function accompanied by frailty and ageing-associated diseases. The most broadly and well-studied pathways influencing ageing are the insulin/insulin-like growth factor 1 signaling pathway and the dietary restriction pathway. Recent studies in diverse organisms have also delineated emerging pathways, which collectively or independently contribute to ageing. Among them the proteostatic-stress-response networks, inextricably affect normal ageing by maintaining or restoring protein homeostasis to preserve proper cellular and organismal function. In this chapter, we survey the involvement of heat stress and endoplasmic reticulum stress responses in the regulation of longevity, placing emphasis on the cross talk between different response mechanisms and their systemic effects. We further discuss novel insights relevant to the molecular pathways mediating these stress responses that may facilitate the development of innovative interventions targeting age-related pathologies such as diabetes, cancer, cardiovascular and neurodegenerative diseases.

  20. Heterologous protein production using the twin arginine translocation pathway

    DOEpatents

    Pohlschroder, Mechtild; Kissinger, Jessica C; Rose, R. Wesley; Brueser, Thomas; Dilks, Kieran

    2008-11-04

    Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

  1. The mevalonate pathway regulates primitive streak formation via protein farnesylation

    PubMed Central

    Okamoto-Uchida, Yoshimi; Yu, Ruoxing; Miyamura, Norio; Arima, Norie; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Yoshida, Suguru; Hosoya, Takamitsu; Nawa, Makiko; Kasama, Takeshi; Asaoka, Yoichi; Alois, Reiner Wimmer; Elling, Ulrich; Penninger, Josef M.; Nishina, Sachiko; Azuma, Noriyuki; Nishina, Hiroshi

    2016-01-01

    The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation. PMID:27883036

  2. Amyloid precursor protein modulates β-catenin degradation

    PubMed Central

    Chen, Yuzhi; Bodles, Angela M

    2007-01-01

    Background The amyloid precursor protein (APP) is genetically associated with Alzheimer's disease (AD). Elucidating the function of APP should help understand AD pathogenesis and provide insights into therapeutic designs against this devastating neurodegenerative disease. Results We demonstrate that APP expression in primary neurons induces β-catenin phosphorylation at Ser33, Ser37, and Thr41 (S33/37/T41) residues, which is a prerequisite for β-catenin ubiquitinylation and proteasomal degradation. APP-induced phosphorylation of β-catenin resulted in the reduction of total β-catenin levels, suggesting that APP expression promotes β-catenin degradation. In contrast, treatment of neurons with APP siRNAs increased total β-catenin levels and decreased β-catenin phosphorylation at residues S33/37/T41. Further, β-catenin was dramatically increased in hippocampal CA1 pyramidal cells from APP knockout animals. Acute expression of wild type APP or of familial AD APP mutants in primary neurons downregulated β-catenin in membrane and cytosolic fractions, and did not appear to affect nuclear β-catenin or β-catenin-dependent transcription. Conversely, in APP knockout CA1 pyramidal cells, accumulation of β-catenin was associated with the upregulation of cyclin D1, a downstream target of β-catenin signaling. Together, these data establish that APP downregulates β-catenin and suggest a role for APP in sustaining neuronal function by preventing cell cycle reactivation and maintaining synaptic integrity. Conclusion We have provided strong evidence that APP modulates β-catenin degradation in vitro and in vivo. Future studies may investigate whether APP processing is necessary for β-catenin downregulation, and determine if excessive APP expression contributes to AD pathogenesis through abnormal β-catenin downregulation. PMID:18070361

  3. Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate.

    PubMed Central

    Oltmanns, R H; Müller, R; Otto, M K; Lingens, F

    1989-01-01

    Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate. PMID:2604392

  4. Breakdown products on metabolic pathway of degradation of benz[a]anthracene by a ligninolytic fungus.

    PubMed

    Cajthaml, Tomás; Erbanová, Pavla; Sasek, Václav; Moeder, Monika

    2006-07-01

    Cultures of the ligninolytic fungus Irpex lacteus incubated in a nutrient liquid medium degraded more than 70% of the initially applied benz[a]anthracene within 14 days. At the first step of metabolization, benz[a]anthracene was transformed via a typical pathway of ligninolytic fungi to benz[a]anthracene-7,12-dione (BaAQ). The product was further transformed by at least two ways, whereas one is complied with the anthracene metabolic pathway of I. lacteus. Benz[a]anthracene-7,12-dione was degraded to 1,2-naphthalenedicarboxylic acid and phthalic acid that was followed with production of 2-hydroxymethyl benzoic acid or monomethyl and dimethylesters of phthalic acid. Another degradation product of BaAQ was identified as 1-tetralone. Its transformation via 1,4-naphthalenedione, 1,4-naphthalenediol and 1,2,3,4-tetrahydro-1-hydroxynaphthalene resulted again in phthalic acid. None of the intermediates were identified as dead-end metabolites. Metabolites produced by ring cleavage of benz[a]anthracene using the ligninolytic fungus are firstly presented in this work.

  5. Arginine deiminase pathway genes and arginine degradation variability in Oenococcus oeni strains.

    PubMed

    Araque, Isabel; Gil, Joana; Carreté, Ramon; Constantí, Magda; Bordons, Albert; Reguant, Cristina

    2016-03-01

    Trace amounts of the carcinogenic ethyl carbamate can appear in wine as a result of a reaction between ethanol and citrulline, which is produced from arginine degradation by some bacteria used in winemaking. In this study, arginine deiminase (ADI) pathway genes were evaluated in 44 Oenococcus oeni strains from wines originating from several locations in order to establish the relationship between the ability of a strain to degrade arginine and the presence of related genes. To detect the presence of arc genes of the ADI pathway in O. oeni, pairs of primers were designed to amplify arcA, arcB, arcC and arcD1 sequences. All strains contained these four genes. The same primers were used to confirm the organization of these genes in an arcABCD1 operon. Nevertheless, considerable variability in the ability to degrade arginine among these O. oeni strains was observed. Therefore, despite the presence of the arc genes in all strains, the expression patterns of individual genes must be strain dependent and influenced by the different wine conditions. Additionally, the presence of arc genes was also determined in the 57 sequenced strains of O. oeni available in GenBank, and the complete operon was found in 83% of strains derived from wine. The other strains were found to lack the arcB, arcC and arcD genes, but all contained sequences homologous to arcA, and some of them had also ADI activity.

  6. Endothelin-converting enzymes degrade intracellular β-amyloid produced within the endosomal/lysosomal pathway and autophagosomes.

    PubMed

    Pacheco-Quinto, Javier; Eckman, Elizabeth A

    2013-02-22

    Impairments in Aβ removal are increasingly being considered as a possible cause for the abnormal Aβ build-up typical of Alzheimer disease. Of particular interest is a pool of Aβ that accumulates intraneuronally and may contribute to neuronal toxicity. The mechanism for intraneuronal accumulation, however, is not well understood and is commonly attributed to impaired removal of extracellular Aβ by neurons. Based on the intracellular distribution of the well established Aβ degrading enzymes, ECE-1 and ECE-2, we tested whether impairments in their catalytic activity could lead to intracellular Aβ accumulation. Using SH-SY5Y cells overexpressing wild-type amyloid precursor protein and pharmacological inhibition of endogenous ECE activity, we found that ECEs participate in the degradation of at least two distinct pools of Aβ; one destined for secretion and the other being produced and degraded within the endosomal-autophagic-lysosomal pathways. Although ECE-1 regulates both pools of Aβ, ECE-2 regulates mainly the intracellular pool of the peptide. Consistent with this result, ECE-2 was found to co-localize with markers of the endosomal/lysosomal pathway but not with a trans-Golgi network marker. Furthermore, ECE-2 was detected in autophagic vesicles in cells treated with chloroquine. Under these conditions, ECE inhibition produced significantly higher elevations in intracellular Aβ than chloroquine treatment alone. This study highlights the existence of Aβ clearance mechanisms by ECEs at intracellular sites of production. Alterations in ECE activity may be considered as a cause for increased intraneuronal Aβ in Alzheimer disease.

  7. (Tri)butyltin biotic degradation rates and pathways in different compartments of a freshwater model ecosystem.

    PubMed

    Tessier, Emmanuel; Amouroux, David; Morin, Anne; Christian, Lehnhoff; Thybaud, Eric; Vindimian, Eric; Donard, Olivier F X

    2007-12-15

    Experiments were conducted in controlled temperate freshwater ecosystems (microcosms) to determine the persistence and biogeochemical dynamic of tributyltin (TBT) and its degradation products. TBT and its derivatives were monitored simultaneously for 23 days (552 h) in sediment-water systems, with or without macroorganisms (macrophytes: Elodea canadensis and gastropods: Lymnaea stagnalis). Biphasic TBT removal from the water column was significantly enhanced by the presence of biota. The persistence of TBT in biota was assessed by a kinetic approach of the different bioaccumulation pathways and associated metabolisms adopted by the snails and the macrophytes in response to the TBT contamination. Furthermore, sediment acted for the final sink for butyltins in both types of microcosms, with more than 70% of TBT and its metabolites recovered in this compartment after two weeks of exposure. Degradation pathways in sediments of both biotic and abiotic microcosms appeared to represent a key process in TBT cycle and were characterized by half-lives in the range of one month. Specific transformation and transfer pathways of TBT as reactional mechanisms are discussed and modelled assessing in detail the role of each compartment with regards to the fate of TBT in the model aquatic ecosystems.

  8. Titanium dioxide-mediated heterogeneous photocatalytic degradation of terbufos: parameter study and reaction pathways.

    PubMed

    Wu, Ren-Jang; Chen, Chiing-Chang; Chen, Ming-Hung; Lu, Chung-Shin

    2009-03-15

    The photocatalytic degradation of terbufos in aqueous suspensions was investigated by using titanium dioxide (TiO(2)) as a photocatalyst. About 99% of terbufos was degraded after UV irradiation for 90 min. Factors such as pH of the system, TiO(2) dosage, and presence of anions were found to influence the degradation rate. Photodegradation of terbufos by TiO(2)/UV exhibited pseudo-first-order reaction kinetics, and a reaction quantum yield of 0.289. The electrical energy consumption per order of magnitude for photocatalytic degradation of terbufos was calculated and showed that a moderated efficiency (E(EO)=71 kWh/(m(3)order)) was obtained in TiO(2)/UV process. To obtain a better understanding of the mechanistic details of this TiO(2)-assisted photodegradation of terbufos with UV irradiation, the intermediates of the processes were separated, identified, and characterized by the solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) technique. The probable photodegradation pathways were proposed and discussed.

  9. Chemical modification and degradation of atrazine in Medicago sativa through multiple pathways.

    PubMed

    Zhang, Jing Jing; Lu, Yi Chen; Yang, Hong

    2014-10-08

    Atrazine is a member of the triazine herbicide family intensively used to control weeds for crop production. In this study, atrazine residues and its degraded products in alfalfa (Medicago sativa) were characterized using UPLC-TOF-MS/MS. Most of atrazine absorbed in plants was found as chemically modified derivatives like deisopropylated atrazine (DIA), dehydrogenated atrazine (DHA), or methylated atrazine (MEA), and some atrazine derivatives were conjugated through different functional groups such as sugar, glutathione, and amino acids. Interestingly, the specific conjugates DHA+hGSH (homoglutathione) and MEA-HCl+hGSH in alfalfa were detected. These results suggest that atrazine in alfalfa can be degraded through different pathways. The increased activities of glycosyltransferase and glutathione S-transferase were determined to support the atrazine degradation models. The outcome of the work uncovered the detailed mechanism for the residual atrazine accumulation and degradation in alfalfa and will help to evaluate whether the crop is suitable to be cultivated in the atrazine-polluted soil.

  10. [A novel biological pathway expansion method based on the knowledge of protein-protein interactions].

    PubMed

    Zhao, Xiaolei; Zuo, Xiaoyu; Qin, Jiheng; Liang, Yan; Zhang, Naizun; Luan, Yizhao; Rao, Shaoqi

    2014-04-01

    Biological pathways have been widely used in gene function studies; however, the current knowledge for biological pathways is per se incomplete and has to be further expanded. Bioinformatics prediction provides us a cheap but effective way for pathway expansion. Here, we proposed a novel method for biological pathway prediction, by intergrating prior knowledge of protein?protein interactions and Gene Ontology (GO) database. First, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to which the interacting neighbors of a targe gene (at the level of protein?protein interaction) belong were chosen as the candidate pathways. Then, the pathways to which the target gene belong were determined by testing whether the genes in the candidate pathways were enriched in the GO terms to which the target gene were annotated. The protein?protein interaction data obtained from the Human Protein Reference Database (HPRD) and Biological General Repository for Interaction Datasets (BioGRID) were respectively used to predict the pathway attribution(s) of the target gene. The results demanstrated that both the average accuracy (the ratio of the correctly predicted pathways to the totally pathways to which all the target genes were annotated) and the relative accuracy (of the genes with at least one annotated pathway being successful predicted, the percentage of the genes with all the annotated pathways being correctly predicted) for pathway predictions were increased with the number of the interacting neighbours. When the number of interacting neighbours reached 22, the average accuracy was 96.2% (HPRD) and 96.3% (BioGRID), respectively, and the relative accuracy was 93.3% (HPRD) and 84.1% (BioGRID), respectively. Further validation analysis of 89 genes whose pathway knowledge was updated in a new database release indicated that 50 genes were correctly predicted for at least one updated pathway, and 43 genes were accurately predicted for all the updated pathways, giving an

  11. Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts

    PubMed Central

    Kmiec, Beata; Teixeira, Pedro F.; Berntsson, Ronnie P.-A.; Murcha, Monika W.; Branca, Rui M. M.; Radomiljac, Jordan D.; Regberg, Jakob; Svensson, Linda M.; Bakali, Amin; Langel, Ülo; Lehtiö, Janne; Whelan, James; Stenmark, Pål; Glaser, Elzbieta

    2013-01-01

    Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8–1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å3. The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts. PMID:24043784

  12. Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

    PubMed

    Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

    2014-05-01

    The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes.

  13. Activation of the S-Phase Checkpoint Inhibits Degradation of the F-Box Protein Dia2 ▿

    PubMed Central

    Kile, Andrew C.; Koepp, Deanna M.

    2010-01-01

    A stable genome is critical to cell viability and proliferation. During DNA replication, the S-phase checkpoint pathway responds to replication stress. In budding yeast, the chromatin-bound F-box protein Dia2 is required to maintain genomic stability and may help replication complexes overcome sites of damaged DNA and natural fragile regions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases. We show here that Dia2 is itself targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint pathway inhibits Dia2 protein degradation. S-phase checkpoint mutants fail to stabilize Dia2 in response to replication stress. Deletion of DIA2 from these checkpoint mutants exacerbates their sensitivity to hydroxyurea, suggesting that stabilization of Dia2 contributes to the replication stress response. Unlike the case for other F-box proteins, deletion of the F-box domain in Dia2 does not stabilize the protein. Rather, an N-terminal domain that is also required for nuclear localization is necessary for degradation. When a strong nuclear localization signal (NLS) is added to dia2 mutants lacking this domain, the Dia2 protein is both stable and nuclear. Together, our results suggest that Dia2 protein turnover does not involve an autocatalytic mechanism and that Dia2 proteolysis is inhibited by activation of the replication stress response. PMID:19858292

  14. Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

    PubMed Central

    Scott, Daniel C.; Schekman, Randy

    2008-01-01

    Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin–proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p. PMID:18573918

  15. Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone.

    PubMed

    Choe, Jong Kwon; Richards, David H; Wilson, Corey J; Mitch, William A

    2015-11-17

    Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation.

  16. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    NASA Astrophysics Data System (ADS)

    Shah, Dhiral Ashwin

    functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. The same concept of naturally occurring protein-protein interactions can also be implemented to selectively bring intracellular protein targets in close proximity to proteasomal degradation machinery in cells and effect their depletion from the cellular compartments. This approach will be able to not only target entire pool of proteins to ubiquitination-mediated degradation, but also to specific sub-pools of posttranslationally modified proteins in the cell, provided peptides having distinct binding affinities are identified for posttranslational modifications. This system can then be tested for intracellular protein delivery using nanoparticle carriers to identify roles of different posttranslational modifications on the protein's activity. In future work, we propose to develop a cellular detection system, based on GFP complementation, which can be used to evaluate the efficiency of different protein delivery carriers to internalize proteins into the cell cytosol. We envision the application of nanoscale materials as intracellular protein delivery vehicles to target diverse cell signaling pathways at the posttranslational level, and subsequent metabolic manipulation, which may have interesting therapeutic properties and can potentially target stem cell fate.

  17. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

    PubMed Central

    Mehnert, Martin; Sommermeyer, Franziska; Berger, Maren; Kumar Lakshmipathy, Sathish; Gauss, Robert; Aebi, Markus; Jarosch, Ernst; Sommer, Thomas

    2015-01-01

    Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1. PMID:25428985

  18. Genes Related to Mitochondrial Functions, Protein Degradation, and Chromatin Folding Are Differentially Expressed in Lymphomonocytes of Rett Syndrome Patients

    PubMed Central

    Leoni, Guido; Cervellati, Franco; Canali, Raffaella; Cortelazzo, Alessio; De Felice, Claudio; Ciccoli, Lucia; Hayek, Joussef

    2013-01-01

    Rett syndrome (RTT) is mainly caused by mutations in the X-linked methyl-CpG binding protein (MeCP2) gene. By binding to methylated promoters on CpG islands, MeCP2 protein is able to modulate several genes and important cellular pathways. Therefore, mutations in MeCP2 can seriously affect the cellular phenotype. Today, the pathways that MeCP2 mutations are able to affect in RTT are not clear yet. The aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (PBMC) isolated from RTT patients to try to evidence new genes and new pathways that are involved in RTT pathophysiology. LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses on microarray data from 12 RTT patients and 7 control subjects identified 482 genes modulated in RTT, of which 430 were upregulated and 52 were downregulated. Functional clustering of a total of 146 genes in RTT identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, RNA processing, and chromatin folding. Our microarray data reveal an overexpression of genes involved in ATP synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. In conclusion, these findings suggest that mitochondrial-ATP-proteasome functions are likely to be involved in RTT clinical features. PMID:24453408

  19. Yarrowia lipolytica vesicle-mediated protein transport pathways

    PubMed Central

    Swennen, Dominique; Beckerich, Jean-Marie

    2007-01-01

    Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y

  20. Methyl-mercury degradation pathways: A comparison among three mercury impacted ecosystems

    USGS Publications Warehouse

    Marvin-DiPasquale, M.; Agee, J.; Mcgowan, C.; Oremland, R.S.; Thomas, M.; Krabbenhoft, D.; Gilmour, C.C.

    2000-01-01

    We examined microbial methylmercury (MeHg) degradation in sediment of the Florida Everglades, Carson River (NV), and San Carlos Creek (CA), three freshwater environments that differ in the extent and type of mercury contamination and sediment biogeochemistry. Degradation rate constant (k(deg)) values increased with total mercury (Hg(t)) contamination both among and within ecosystems. The highest k(deg)'s (2.8-5.8 d-1) were observed in San Carlos Creek, at acid mine drainage impacted sites immediately downstream of the former New Idria mercury mine, where Hg(t) ranged from 4.5 to 21.3 ppm (dry wt). A reductive degradation pathway (presumably mer-detoxification) dominated degradation at these sites, as indicated by the nearly exclusive production of 14CH4 from 14C-MeHg, under both aerobic and anaerobic conditions. At the upstream control site, and in the less contaminated ecosystems (e.g. the Everglades), k(deg)'s were low (???0.2 d-1) and oxidative demethylation (OD) dominated degradation, as evident from 14CO2 production. k(deg) increased with microbial CH4 production, organic content, and reduced sulfur in the Carson River system and increased with decreasing pH in San Carlos Creek. OD associated CO2 production increased with pore-water SO42- in Everglades samples but was not attributable to anaerobic methane oxidation, as has been previously proposed. This ecosystem comparison indicates that severely contaminated sediments tend to have microbial populations that actively degrade MeHg via mer-detoxification, whereas OD occurs in heavily contaminated sediments as well but dominates in those less contaminated.We examined microbial methylmercury (MeHg) degradation in sediment of the Florida Everglades, Carson River (NV), and San Carlos Creek (CA), three freshwater environments that differ in the extent and type of mercury contamination and sediment biogeochemistry. Degradation rate constant (kdeg) values increased with total mercury (Hgt) contamination both among and

  1. Predominant archaea in marine sediments degrade detrital proteins.

    PubMed

    Lloyd, Karen G; Schreiber, Lars; Petersen, Dorthe G; Kjeldsen, Kasper U; Lever, Mark A; Steen, Andrew D; Stepanauskas, Ramunas; Richter, Michael; Kleindienst, Sara; Lenk, Sabine; Schramm, Andreas; Jørgensen, Bo Barker

    2013-04-11

    Half of the microbial cells in the Earth's oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.

  2. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly.

    PubMed

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D; Aebi, Markus; Gauss, Robert

    2015-07-15

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation.

  3. BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation.

    PubMed

    Swaminathan, Gayathri; Zhu, Wan; Plowey, Edward D

    2016-12-01

    The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267-337). Deletion of a BECN1 ECD subregion (amino acids 285-299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.

  4. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

    PubMed

    Yabe, Ryotaro; Miura, Akane; Usui, Tatsuya; Mudrak, Ingrid; Ogris, Egon; Ohama, Takashi; Sato, Koichi

    2015-01-01

    Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.

  5. Heat-induced Protein Structure and Subfractions in Relation to Protein Degradation Kinetics and Intestinal Availability in Dairy Cattle

    SciTech Connect

    Doiron, K.; Yu, P; McKinnon, J; Christensen, D

    2009-01-01

    The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included (1) protein structure a-helix to e-sheet ratio; (2) protein subfractions profiles; (3) protein degradation kinetics and effective degradability; (4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120C for 40 and 60 min increased the protein structure a-helix to e-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences

  6. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor.

    PubMed

    de Laat, Rian; Meabon, James S; Wiley, Jesse C; Hudson, Mark P; Montine, Thomas J; Bothwell, Mark

    2015-01-01

    Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP) by β-secretase and γ-secretase generate amyloid β (Aβ) peptides, which are thought to contribute to Alzheimer's disease (AD). Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

  7. VCP cooperates with UBXD1 to degrade mitochondrial outer membrane protein MCL1 in model of Huntington's disease.

    PubMed

    Guo, Xing; Qi, Xin

    2017-02-01

    Proteasome-dependent turnover of mitochondrial outer membrane (OMM)-associated proteins is one of the mechanisms for maintaining proper mitochondrial quality and function. However, the underlying pathways and their implications in human disease are poorly understood. Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by expanded CAG repeats in the N terminal of the huntingtin gene (mutant Huntingtin, mtHtt). In this study, we show an extensive degradation of the OMM protein MCL1 (Myeloid cell leukemia sequence 1) in both HD mouse striatal cells and HD patient fibroblasts. The decrease in MCL1 level is associated with mitochondrial and cellular damage. Valosin-containing-protein (VCP) is an AAA-ATPase central to protein turnover via the ubiquitin proteasome system (UPS). We found that VCP translocates to mitochondria and promotes MCL1 degradation in HD cell cultures. Either down-regulation of VCP by RNA interference or inhibition of VCP by a dominant negative mutant abolishes MCL1 degradation in HD cell cultures. We further show that UBX-domain containing protein 1 (UBXD1), a known co-factor of VCP assisting in the recognition of substrates for protein degradation, selectively binds to MCL1 and interacts with VCP to mediate MCL1 extraction from the mitochondria. These results indicate that the OMM protein MCL1 is degraded by the VCP-UBXD1 complex and that the process is promoted by the presence of mtHtt. Therefore, our finding provides a new insight into the mechanism of mitochondrial dysfunction in HD.

  8. Mapping protein electron transfer pathways with QM/MM methods

    PubMed Central

    Guallar, Victor; Wallrapp, Frank

    2008-01-01

    Mixed quantum mechanics/molecular mechanics (QM/MM) methods offer a valuable computational tool for understanding the electron transfer pathway in protein–substrate interactions and protein–protein complexes. These hybrid methods are capable of solving the Schrödinger equation on a small subset of the protein, the quantum region, describing its electronic structure under the polarization effects of the remainder of the protein. By selectively turning on and off different residues in the quantum region, we are able to obtain the electron pathway for short- and large-range interactions. Here, we summarize recent studies involving the protein–substrate interaction in cytochrome P450 camphor, ascorbate peroxidase and cytochrome c peroxidase, and propose a novel approach for the long-range protein–protein electron transfer. The results on ascorbate peroxidase and cytochrome c peroxidase reveal the importance of the propionate groups in the electron transfer pathway. The long-range protein–protein electron transfer has been studied on the cytochrome c peroxidase–cytochrome c complex. The results indicate the importance of Phe82 and Cys81 on cytochrome c, and of Asn196, Ala194, Ala176 and His175 on cytochrome c peroxidase. PMID:18445553

  9. Kinetics and pathways of ibuprofen degradation by the UV/chlorine advanced oxidation process.

    PubMed

    Xiang, Yingying; Fang, Jingyun; Shang, Chii

    2016-03-01

    The UV/chlorine advanced oxidation process (AOP), which forms reactive species such as hydroxyl radicals (HO) and reactive chlorine species (RCS) such as chlorine atoms (Cl) and Cl2(-), is being considered as an alternative to the UV/H2O2 AOP for the degradation of emerging contaminants. This study investigated the kinetics and pathways of the degradation of a recalcitrant pharmaceutical and personal care product (PPCP)-ibuprofen (IBP)-by the UV/chlorine AOP. The degradation of IBP followed the pseudo first-order kinetics. The first-order rate constant was 3.3 times higher in the UV/chlorine AOP than in the UV/H2O2 AOP for a given chemical molar dosage at pH 6. The first-order rate constant decreased from 3.1 × 10(-3) s(-1) to 5.5 × 10(-4) s(-1) with increasing pH from 6 to 9. Both HO and RCS contributed to the degradation, and the contribution of RCS increased from 22% to 30% with increasing pH from 6 to 9. The degradation was initiated by HO-induced hydroxylation and Cl-induced chlorine substitution, and sustained through decarboxylation, demethylation, chlorination and ring cleavage to form more stable products. Significant amounts of chlorinated intermediates/byproducts were formed from the UV/chlorine AOP, and four chlorinated products were newly identified. The yield of total organic chlorine (TOCl) was 31.6 μM after 90% degradation of 50 μM IBP under the experimental conditions. The known disinfection by-products (DBPs) comprised 17.4% of the TOCl. The effects of water matrix in filtered drinking water on the degradation were not significant, demonstrating the practicality of the UV/chlorine AOP for the control of some refractory PPCPs. However, the toxicity of the chlorinated products should be further assessed.

  10. Kinetics and reaction pathways of formaldehyde degradation using the UV-fenton method.

    PubMed

    Liu, Xiangxuan; Liang, Jiantao; Wang, Xuanjun

    2011-05-01

    This study was based on the purpose of investigating the reaction rules of formaldehyde (HCHO) as an intermediate product in the degradation of many other organic wastewaters. The process conditions of UV-Fenton method for the degradation of the low concentrations of HCHO were studied in a batch photochemical reactor. The results showed that, when the original HCHO concentration was 30 mg/L, at an operating temperature of 23 degrees C, pH = 3, an H202 dosage of 68 mg/L, and an H2O2-to-Fe2+ mole ratio (H2O2:Fe2+) of 5, 91.89% of the HCHO was removed after 30 minutes. The degradation of HCHO in the UV-Fenton system was basically in accordance with the exponential decay. The kinetic study results showed that the reaction orders of HCHO, Fe2+, and H2O2 in the system were 1.054, 0.510, and 0.728, respectively, and the activation energy (Ea) was 9.85 kJ/mol. The comparison of UV/H2O2, Fenton, and UV-Fenton systems for the degradation of HCHO, and the results of iron catalyst tests showed that the mechanism of UV-Fenton on the degradation of HCHO was through a synergistic effect of Fe2+ and UV light to catalyze the decomposition of H2O2. The introduction of UV irradiation to the Fenton system largely increased the degradation rate of HCHO, mainly as a result of the accelerating effect on the formation of the Fe2+/Fe3+ cycle. The reaction products were analyzed by gas chromatography-mass spectrometry and a chemical oxygen demand (COD) analyzer. The effluent gases also were analyzed by gas chromatography. Based on those results, the reaction pathways of HCHO in the UV-Fenton system were proposed. The qualitative and quantitative analysis of the reaction products and the COD showed that the main intermediate product of the reaction was formic acid, and the further oxidation of it was the rate-limiting step for the degradation of HCHO.

  11. Pathways and determinants of early spontaneous vegetation succession in degraded lowland of South China.

    PubMed

    Duan, Wen-Jun; Ren, Hai; Fu, Sheng-Lei; Guo, Qin-Feng; Wang, Jun

    2008-02-01

    Continuous and prolonged human disturbances have caused severe degradation of a large portion of lowland in South China, and how to restore such degraded ecosystems becomes an increasing concern. The process and mechanisms of spontaneous succession, which plays an important role in vegetation restoration, have not been adequately examined. To identify the pathways of early spontaneous vegetation succession, 41 plots representing plant communities abandoned over different times were established and investigated. The communities and indicator species of the vegetation were classified by analyzing the important values of plant species using multivariate analyses. The results indicated that the plant species could be classified into nine plant communities representing six succession stages. The pathway and species composition also changed in the process of succession. We also measured 13 environmental variables of microtopography, soil structure and soil nutrition in each plot to examine the driving forces of succession and the vegetation-environment relationships. Our results showed that the environmental variables changed in diverse directions, and that soil bulk density, soil water capacity and soil acidity were the most important factors.

  12. Genomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway.

    PubMed

    Foster, Alexander; Barnes, Nicole; Speight, Robert; Keane, Mark A

    2013-10-01

    The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis-Menten kinetic analysis delivered a Michaelis constant (K(M)=0.014 mM) and maximum rate (Vmax=11.2 μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value=0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria.

  13. Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

    PubMed Central

    Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

    2010-01-01

    Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation. PMID:20478262

  14. Selective STAT protein degradation induced by paramyxoviruses requires both STAT1 and STAT2 but is independent of alpha/beta interferon signal transduction.

    PubMed

    Parisien, Jean-Patrick; Lau, Joe F; Rodriguez, Jason J; Ulane, Christina M; Horvath, Curt M

    2002-05-01

    The alpha/beta interferon (IFN-alpha/beta)-induced STAT signal transduction pathway leading to activation of the ISGF3 transcription complex and subsequent antiviral responses is the target of viral pathogenesis strategies. Members of the Rubulavirus genus of the Paramyxovirus family of RNA viruses have acquired the ability to specifically target either STAT1 or STAT2 for proteolytic degradation as a countermeasure for evading IFN responses. While type II human parainfluenza virus induces STAT2 degradation, simian virus 5 induces STAT1 degradation. The components of the IFN signaling system that are required for STAT protein degradation by these paramyxoviruses have been investigated in a series of human somatic cell lines deficient in IFN signaling proteins. Results indicate that neither the IFN-alpha/beta receptor, the tyrosine kinases Jak1 or Tyk2, nor the ISGF3 DNA-binding subunit, IFN regulatory factor 9 (IRF9), is required for STAT protein degradation induced by either virus. Nonetheless, both STAT1 and STAT2 are strictly required in the host cell to establish a degradation-permissive environment enabling both viruses to target their respective STAT protein. Complementation studies reveal that STAT protein-activating tyrosine phosphorylation and functional src homology 2 (SH2) domains are dispensable for creating a permissive STAT degradation environment in degradation-incompetent cells, but the N terminus of the missing STAT protein is essential. Protein-protein interaction analysis indicates that V and STAT proteins interact physically in vitro and in vivo. These results constitute genetic and biochemical evidence supporting a virus-induced, IFN-independent STAT protein degradation complex that contains at least STAT1 and STAT2.

  15. Machine Learning of Protein Interactions in Fungal Secretory Pathways.

    PubMed

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja; Rousu, Juho

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker's yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities.

  16. Machine Learning of Protein Interactions in Fungal Secretory Pathways

    PubMed Central

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker’s yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities. PMID:27441920

  17. Paeoniflorin inhibits human glioma cells via STAT3 degradation by the ubiquitin–proteasome pathway

    PubMed Central

    Nie, Xiao-hu; Ou-yang, Jia; Xing, Ying; Li, Dan-yan; Dong, Xing-yu; Liu, Ru-en; Xu, Ru-xiang

    2015-01-01

    We investigated the underlying mechanism for the potent proapoptotic effect of paeoniflorin (PF) on human glioma cells in vitro, focusing on signal transducer and activator of transcription 3 (STAT3) signaling. Significant time- and dose-dependent apoptosis and inhibition of proliferation were observed in PF-treated U87 and U251 glioma cells. Expression of STAT3, its active form phosphorylated STAT3 (p-STAT3), and several downstream molecules, including HIAP, Bcl-2, cyclin D1, and Survivin, were significantly downregulated upon PF treatment. Overexpression of STAT3 induced resistance to PF, suggesting that STAT3 was a critical target of PF. Interestingly, rapid downregulation of STAT3 was consistent with its accelerated degradation, but not with its dephosphorylation or transcriptional modulation. Using specific inhibitors, we demonstrated that the prodegradation effect of PF on STAT3 was mainly through the ubiquitin–proteasome pathway rather than via lysosomal degradation. These findings indicated that PF-induced growth suppression and apoptosis in human glioma cells through the proteasome-dependent degradation of STAT3. PMID:26508835

  18. Photocatalytic transformation of sixteen substituted phenylurea herbicides in aqueous semiconductor suspensions: intermediates and degradation pathways.

    PubMed

    Fenoll, José; Sabater, Paula; Navarro, Gines; Pérez-Lucas, Gabriel; Navarro, Simón

    2013-01-15

    The photocatalytic degradation of sixteen substituted phenylurea herbicides (PUHs) in pure water has been studied using zinc oxide (ZnO) and titanium dioxide (TiO(2)) as photocatalyst under artificial light irradiation. Photocatalytic experiments showed that the addition of these chalcogenide oxides in tandem with the oxidant (Na(2)S(2)O(8)) strongly enhances the degradation rate of these compounds in comparison with those carried out with ZnO and TiO(2) alone and photolytic tests. Comparison of catalysts showed that ZnO is the most efficient for the removal of such herbicides in optimal conditions and at constant volumetric rate of photon absorption in the photoreactor. Thus, the complete disappearance of all the studied compounds was achieved after 20 min of illumination in the ZnO/Na(2)S(2)O(8) system. The main photocatalytic intermediates detected during the degradation of PUHs were identified. The probable photodegradation pathways were proposed and discussed. The main steps involved: N-demethylation of the N,N-dimethylurea-substituted compounds followed of N-demethylation and N-demethoxylation of the N-methoxy-N-methyl-substituted ureas and hydroxylation of aromatic rings and their aliphatic side-chains of both, parent compounds and intermediates.

  19. Aqueous photodegradation of 4-tert-butylphenol: By-products, degradation pathway and theoretical calculation assessment.

    PubMed

    Wu, Yanlin; Shi, Jin; Chen, Hongche; Zhao, Jianfu; Dong, Wenbo

    2016-10-01

    4-tert-butylphenol (4-t-BP), an endocrine disrupting chemical, is widely distributed in natural bodies of water but is difficult to biodegrade. In this study, we focused on the transformation of 4-t-BP in photo-initiated degradation processes. The steady-state photolysis and laser flash photolysis (LFP) experiments were conducted in order to elucidate its degradation mechanism. Identification of products was performed using the GC-MS, LC-MS and theoretical calculation techniques. The oxidation pathway of 4-t-BP by hydroxyl radical (HO) was also studied and H2O2 was added to produce HO. 4-tert-butylcatechol and 4-tert-butylphenol dimer were produced in 4-t-BP direct photolysis. 4-tert-butylcatechol and hydroquinone were produced by the oxidation of HO. But the formation mechanism of 4-tert-butylcatechol in the two processes was different. The benzene ring was fractured in 4-t-BP oxidation process and 29% of TOC was degraded after 16h irradiation.

  20. Degradation Pathway for Eplerenone by Validated Stability Indicating UP-LC Method.

    PubMed

    Sudhakar Babu, Kondru; Madireddy, Venkataramanna; Indukuri, Venkata Somaraju

    2012-01-01

    Degradation pathway for eplerenone is established as per ICH recommendations by validated and stability-indicating reverse phase liquid chromatographic method. Eplerenone is subjected to stress conditions of acid, base, oxidation, and thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Four impurities are studied and the major degradant (RRT about 0.31) was identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Efficient chromatographic separation is achieved on a Waters symmetry C18 stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 240 nm at a flow rate of 1.0 mL min(-1). In the developed LC method the resolution between eplerenone and four potential impurities (imp-1, imp-2, imp-3, and imp-4) is found to be greater than 4.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for eplerenone and four potential impurities. This method is capable to detect the impurities of eplerenone at a level of 0.020% with respect to test concentration of 1.0 mg mL(-1) for a 20 μL injection volume. The developed UPLC method is validated with respect to specificity, linearity and range, accuracy, precision, and robustness for impurities and assay determination.

  1. Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

    PubMed Central

    Grifoll, M; Selifonov, S A; Chapman, P J

    1994-01-01

    A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed. PMID:8074523

  2. Lactational response of dairy cows to change of degradability of dietary protein and organic matter.

    PubMed

    Aharoni, Y; Arieli, A; Tagari, H

    1993-11-01

    The effect of variable degradability of both OM and CP, incorporated at a constant ratio in diets of high yielding dairy cows (35 kg/d), was studied under commercial dairy herd conditions. Two diets containing 17% CP were formulated, including high (70%) and low (65%) protein degradability. The ratio of rumen-degradable OM to degradable protein was adjusted to 5:1 in both diets. Cows were assigned to treatments based on equal milk yield prior to trial, parity, and DIM. The trial lasted 7 wk: a reference week (wk 0), in which both groups were fed the high degradability diet, was followed by 6 experimental wk, in which group 1 was fed the high degradability diet and group 2 the low degradability diet. Cows on the low degradability diet consumed 1.2 kg more DM and yielded 1.5 kg/d more milk, .055 kg/d more milk protein, and .196 kg/d more milk fat. Percentages of milk protein (3.06 and 3.03) were similar, but fat (3.67 and 3.28) was higher for cows fed the low degradability diet. The results suggest that, when diets were formulated to balance rumen degradability of both OM and CP, 65% rather than 70% degradability of CP was advantageous for yields of milk and milk components.

  3. AMP-activated protein kinase activation leads to lysome-mediated NA(+)/I(-)-symporter protein degradation in rat thyroid cells.

    PubMed

    Cazarin, J M; Andrade, B M; Carvalho, D P

    2014-05-01

    Iodide uptake by thyroid cells is mediated by a transmembrane glycoprotein known as the Na+/I--symporter (NIS). NIS-mediated iodide uptake plays important physiological role in thyroid gland function, as well as in diagnostic and treatment of Graves' disease and thyroid cancer. Although different studies investigated the transcriptional mechanisms of NIS expression, there is no report on the NIS post-translational regulation related to NIS protein degradation in thyroid cells. Recently, our group showed that AMP-activated protein kinase (AMPK) plays a pivotal role in the rat thyroid gland, downregulating iodide uptake, NIS protein, and mRNA content. Since several studies demonstrated that AMPK regulates post-transcriptional mechanisms, such as autophagy-mediated processes in different tissues, we hypothesized that AMPK activation could also regulate NIS protein degradation through the lysosome pathway in thyroid cells. Rat follicular thyroid PCCL3 cells cultivated in Ham's F12 supplemented with 5% calf serum and hormones were exposed to the AMPK pharmacological activator 5-aminoimidazole-4 carboxamide ribonucleoside (AICAR), in the presence or absence of Bafilomycin A1 or MG132 for 24 h. Treatment of PCCL3 cells with Bafilomycin A1 fully prevented the decrease of iodide uptake and NIS protein content mediated by AMPK activation. In contrast, the treatment with MG132 was unable to prevent the effects of AMPK activation on NIS. Our results show that AMPK activation significantly induces NIS protein degradation through a lysosome-mediated mechanism.

  4. Glucosidase II and MRH-domain containing proteins in the secretory pathway

    PubMed Central

    D’Alessio, Cecilia; Dahms, Nancy M.

    2015-01-01

    N -glycosylation in the endoplasmic reticulum (ER) consists of the transfer of a pre-assembled glycan conserved among species (Glc3Man9GlcNAc2) from a lipid donor to a consensus sequence within a nascent protein that is entering the ER. The protein-linked glycans are then processed by glycosidases and glycosyltransferases in the ER producing specific structures that serve as signalling molecules for the fate of the folding glycoprotein: to stay in the ER during the folding process, to be retrotranslocated to the cytosol for proteasomal degradation if irreversibly misfolded, or to pursue transit through the secretory pathway as a mature glycoprotein. In the ER, each glycan signalling structure is recognized by a specific lectin. A domain similar to that of the mannose 6-phosphate receptors (MPRs) has been identified in several proteins of the secretory pathway. These include the beta subunit of glucosidase II (GII), a key enzyme in the early processing of the transferred glycan that removes middle and innermost glucoses and is involved in quality control of glycoprotein folding in the ER (QC), the lectins OS-9 and XTP3-B, proteins involved in the delivery of ER misfolded proteins to degradation (ERAD), the gamma subunit of the Golgi GlcNAc-1-phosphotransferase, an enzyme involved in generating the mannose 6-phosphate (M6P) signal for sorting acidic hydrolases to lysosomes, and finally the MPRs that deliver those hydrolytic enzymes to the lysosome. Each of the MRH-containing proteins recognizes a different signalling N-glycan structure. Three-dimensional structures of some of the MRH domains have been solved, providing the basis to understand recognition mechanisms. PMID:25692846

  5. Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?

    NASA Astrophysics Data System (ADS)

    Scoma, Alberto; Barbato, Marta; Hernandez-Sanabria, Emma; Mapelli, Francesca; Daffonchio, Daniele; Borin, Sara; Boon, Nico

    2016-03-01

    Oil spills represent an overwhelming carbon input to the marine environment that immediately impacts the sea surface ecosystem. Microbial communities degrading the oil fraction that eventually sinks to the seafloor must also deal with hydrostatic pressure, which linearly increases with depth. Piezosensitive hydrocarbonoclastic bacteria are ideal candidates to elucidate impaired pathways following oil spills at low depth. In the present paper, we tested two strains of the ubiquitous Alcanivorax genus, namely A. jadensis KS_339 and A. dieselolei KS_293, which is known to rapidly grow after oil spills. Strains were subjected to atmospheric and mild pressure (0.1, 5 and 10 MPa, corresponding to a depth of 0, 500 and 1000 m, respectively) providing n-dodecane as sole carbon source. Pressures equal to 5 and 10 MPa significantly lowered growth yields of both strains. However, in strain KS_293 grown at 10 MPa CO2 production per cell was not affected, cell integrity was preserved and PO43‑ uptake increased. Analysis of its transcriptome revealed that 95% of its genes were downregulated. Increased transcription involved protein synthesis, energy generation and respiration pathways. Interplay between these factors may play a key role in shaping the structure of microbial communities developed after oil spills at low depth and limit their bioremediation potential.

  6. Sulfoacetate Is Degraded via a Novel Pathway Involving Sulfoacetyl-CoA and Sulfoacetaldehyde in Cupriavidus necator H16

    PubMed Central

    Weinitschke, Sonja; Hollemeyer, Klaus; Kusian, Bernhard; Bowien, Botho; Smits, Theo H. M.; Cook, Alasdair M.

    2010-01-01

    Bacterial degradation of sulfoacetate, a widespread natural product, proceeds via sulfoacetaldehyde and requires a considerable initial energy input. Whereas the fate of sulfoacetaldehyde in Cupriavidus necator (Ralstonia eutropha) H16 is known, the pathway from sulfoacetate to sulfoacetaldehyde is not. The genome sequence of the organism enabled us to hypothesize that the inducible pathway, which initiates sau (sulfoacetate utilization), involved a four-gene cluster (sauRSTU; H16_A2746 to H16_A2749). The sauR gene, divergently orientated to the other three genes, probably encodes the transcriptional regulator of the presumed sauSTU operon, which is subject to inducible transcription. SauU was tentatively identified as a transporter of the major facilitator superfamily, and SauT was deduced to be a sulfoacetate-CoA ligase. SauT was a labile protein, but it could be separated and shown to generate AMP and an unknown, labile CoA-derivative from sulfoacetate, CoA, and ATP. This unknown compound, analyzed by MALDI-TOF-MS, had a relative molecular mass of 889.7, which identified it as protonated sulfoacetyl-CoA (calculated 889.6). SauS was deduced to be sulfoacetaldehyde dehydrogenase (acylating). The enzyme was purified 175-fold to homogeneity and characterized. Peptide mass fingerprinting confirmed the sauS locus (H16_A2747). SauS converted sulfoacetyl-CoA and NADPH to sulfoacetaldehyde, CoA, and NADP+, thus confirming the hypothesis. PMID:20693281

  7. Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?

    PubMed

    Scoma, Alberto; Barbato, Marta; Hernandez-Sanabria, Emma; Mapelli, Francesca; Daffonchio, Daniele; Borin, Sara; Boon, Nico

    2016-03-29

    Oil spills represent an overwhelming carbon input to the marine environment that immediately impacts the sea surface ecosystem. Microbial communities degrading the oil fraction that eventually sinks to the seafloor must also deal with hydrostatic pressure, which linearly increases with depth. Piezosensitive hydrocarbonoclastic bacteria are ideal candidates to elucidate impaired pathways following oil spills at low depth. In the present paper, we tested two strains of the ubiquitous Alcanivorax genus, namely A. jadensis KS_339 and A. dieselolei KS_293, which is known to rapidly grow after oil spills. Strains were subjected to atmospheric and mild pressure (0.1, 5 and 10 MPa, corresponding to a depth of 0, 500 and 1000 m, respectively) providing n-dodecane as sole carbon source. Pressures equal to 5 and 10 MPa significantly lowered growth yields of both strains. However, in strain KS_293 grown at 10 MPa CO2 production per cell was not affected, cell integrity was preserved and PO4(3-) uptake increased. Analysis of its transcriptome revealed that 95% of its genes were downregulated. Increased transcription involved protein synthesis, energy generation and respiration pathways. Interplay between these factors may play a key role in shaping the structure of microbial communities developed after oil spills at low depth and limit their bioremediation potential.

  8. Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?

    PubMed Central

    Scoma, Alberto; Barbato, Marta; Hernandez-Sanabria, Emma; Mapelli, Francesca; Daffonchio, Daniele; Borin, Sara; Boon, Nico

    2016-01-01

    Oil spills represent an overwhelming carbon input to the marine environment that immediately impacts the sea surface ecosystem. Microbial communities degrading the oil fraction that eventually sinks to the seafloor must also deal with hydrostatic pressure, which linearly increases with depth. Piezosensitive hydrocarbonoclastic bacteria are ideal candidates to elucidate impaired pathways following oil spills at low depth. In the present paper, we tested two strains of the ubiquitous Alcanivorax genus, namely A. jadensis KS_339 and A. dieselolei KS_293, which is known to rapidly grow after oil spills. Strains were subjected to atmospheric and mild pressure (0.1, 5 and 10 MPa, corresponding to a depth of 0, 500 and 1000 m, respectively) providing n-dodecane as sole carbon source. Pressures equal to 5 and 10 MPa significantly lowered growth yields of both strains. However, in strain KS_293 grown at 10 MPa CO2 production per cell was not affected, cell integrity was preserved and PO43− uptake increased. Analysis of its transcriptome revealed that 95% of its genes were downregulated. Increased transcription involved protein synthesis, energy generation and respiration pathways. Interplay between these factors may play a key role in shaping the structure of microbial communities developed after oil spills at low depth and limit their bioremediation potential. PMID:27020120

  9. Ubiquitination-mediated degradation of cell cycle-related proteins by F-box proteins.

    PubMed

    Zheng, Nana; Wang, Zhiwei; Wei, Wenyi

    2016-04-01

    F-box proteins, subunits of SKP1-cullin 1-F-box protein (SCF) type of E3 ubiquitin ligase complexes, have been validated to play a crucial role in governing various cellular processes such as cell cycle, cell proliferation, apoptosis, migration, invasion and metastasis. Recently, a wealth of evidence has emerged that F-box proteins is critically involved in tumorigenesis in part through governing the ubiquitination and subsequent degradation of cell cycle proteins, and dysregulation of this process leads to aberrant cell cycle progression and ultimately, tumorigenesis. Therefore, in this review, we describe the critical role of F-box proteins in the timely regulation of cell cycle. Moreover, we discuss how F-box proteins involve in tumorigenesis via targeting cell cycle-related proteins using biochemistry studies, engineered mouse models, and pathological gene alternations. We conclude that inhibitors of F-box proteins could have promising therapeutic potentials in part through controlling of aberrant cell cycle progression for cancer therapies.

  10. Fucoidan inhibition of lung cancer in vivo and in vitro : role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation.

    PubMed

    Hsu, Hsien-Yeh; Lin, Tung-Yi; Wu, Yu-Chung; Tsao, Shu-Ming; Hwang, Pai-An; Shih, Yu-Wei; Hsu, Jason

    2014-09-15

    Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor β (TGFβ) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFβ stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFβ receptors.

  11. Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

    PubMed

    Millership, C; Phillips, J J; Main, E R G

    2016-05-08

    Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch.

  12. Further characterization of o-nitrobenzaldehyde degrading bacterium Pseudomonas sp. ONBA-17 and deduction on its metabolic pathway.

    PubMed

    Yu, Fang-Bo; Li, Xiao-Dan; Ali, Shinawar Waseem; Shan, Sheng-Dao; Luo, Lin-Ping; Guan, Li-Bo

    2014-01-01

    A previously reported o-nitrobenzaldehyde (ONBA) degrading bacterium Pseudomonas sp. ONBA-17 was further identified and characterized. Based on results of DNA base composition and DNA-DNA hybridization, the strain was identified as P. putida. Its degradation effect enhanced with increase of inoculum amount and no lag phase was observed. Higher removal rate was achieved under shaking conditions. All tested ONBA with different initial concentrations could be completely degraded within 5 d. In addition, degradative enzyme(s) involved was confirmed as intra-cellular distributed and constitutively expressed. Effects of different compounds on relative activity of degradative enzyme(s) within cell-free extract were also evaluated. Finally, 2-nitrobenzoic acid and 2, 3-dihydroxybenzoic acid were detected as metabolites of ONBA degradation by P. putida ONBA-17, and relevant metabolic pathway was preliminary proposed. This study might help with future research in better understanding of nitroaromatics biodegradation.

  13. Polymerization of secretory IgM in B lymphocytes is prevented by a prior targeting to a degradation pathway.

    PubMed

    Shachar, I; Amitay, R; Rabinovich, E; Haimovich, J; Bar-Nun, S

    1992-12-05

    B lymphocytes express on their surface a membrane form of IgM (mIgM), and synthesize but fail to secrete a secretory form of IgM (sIgM). Plasma cells shift to the exclusive synthesis and efficient secretion of sIgM. The sIgM in B cells differs from that in plasma cells in its pattern of assembly: in plasma cells, monomers of sIgM are assembled into polymers and only polymers are secreted; in B lymphocytes, monomeric sIgM is neither polymerized nor secreted and is degraded intracellularly. In this article we blocked the export of proteins from the endoplasmic reticulum at low temperatures or with energy poisons or brefeldin A, and localized the different assembly steps of mIgM and sIgM in the 38C B lymphocytes and of sIgM in the 38C-derived sIgM-secreting D2 hybridoma. In both cell lines, sIgM assembly into monomers was not affected, whereas polymerization of sIgM in D2 cells and monomer formation of mIgM in 38C cells were strongly inhibited. Moreover, probing with specific lectins revealed galactosylated monomers and polymers in D2 cells and galactosylated hemimer and monomers only of mIgM in 38C cells. In addition, when Golgi functions were hampered with Tris base, monomerization of mIgM and polymerization of sIgM were attenuated. These results indicate that polymerization of sIgM in D2 cells and monomerization of mIgM in 38C cells are post-endoplasmic reticulum events, occurring in or beyond the trans-Golgi galactosylation compartment. Since only polymers are secreted from D2 cells and only monomeric mIgM is displayed on the surface of 38C cells, partially assembled molecules may traverse the secretory pathway yet are restricted from the cell surface. Furthermore, monomeric sIgM in 38C cells is never galactosylated, thus it is degraded prior to the galactosylation compartment. We conclude that targeting of sIgM to degradation in 38C cells precedes its assembly site into polymers in D2 cells. This implies that degradation of sIgM does not result from the

  14. N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli

    PubMed Central

    Sekar, Karthik; Gentile, Andrew M.; Bostick, John W.; Tyo, Keith E. J.

    2016-01-01

    Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits. PMID:26900850

  15. Wilms Tumor Gene on X Chromosome (WTX) Inhibits Degradation of NRF2 Protein through Competitive Binding to KEAP1 Protein*

    PubMed Central

    Camp, Nathan D.; James, Richard G.; Dawson, David W.; Yan, Feng; Davison, James M.; Houck, Scott A.; Tang, Xiaobo; Zheng, Ning; Major, Michael B.; Moon, Randall T.

    2012-01-01

    WTX is a tumor suppressor protein that is lost or mutated in up to 30% of cases of Wilms tumor. Among its known functions, WTX interacts with the β-transducin repeat containing family of ubiquitin ligase adaptors and promotes the ubiquitination and degradation of the transcription factor β-catenin, a key control point in the WNT/β-catenin signaling pathway. Here, we report that WTX interacts with a second ubiquitin ligase adaptor, KEAP1, which functions to regulate the ubiquitination of the transcription factor NRF2, a key control point in the antioxidant response. Surprisingly, we find that unlike its ability to promote the ubiquitination of β-catenin, WTX inhibits the ubiquitination of NRF2. WTX and NRF2 compete for binding to KEAP1, and thus loss of WTX leads to rapid ubiquitination and degradation of NRF2 and a reduced response to cytotoxic insult. These results expand our understanding of the molecular mechanisms of WTX and reveal a novel regulatory mechanism governing the antioxidant response. PMID:22215675

  16. Degradation of the synthetic dye amaranth by the fungus Bjerkandera adusta Dec 1: inference of the degradation pathway from an analysis of decolorized products.

    PubMed

    Gomi, Nichina; Yoshida, Shuji; Matsumoto, Kazutsugu; Okudomi, Masayuki; Konno, Hiroki; Hisabori, Toru; Sugano, Yasushi

    2011-11-01

    We examined the degradation of amaranth, a representative azo dye, by Bjerkandera adusta Dec 1. The degradation products were analyzed by high performance liquid chromatography (HPLC), visible absorbance, and electrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS). At the primary culture stage (3 days), the probable reaction intermediates were 1-aminonaphthalene-2,3,6-triol, 4-(hydroxyamino) naphthalene-1-ol, and 2-hydroxy-3-[2-(4-sulfophenyl) hydrazinyl] benzenesulfonic acid. After 10 days, the reaction products detected were 4-nitrophenol, phenol, 2-hydroxy-3-nitrobenzenesulfonic acid, 4-nitrobenzene sulfonic acid, and 3,4'-disulfonyl azo benzene, suggesting that no aromatic amines were created. Manganese-dependent peroxidase activity increased sharply after 3 days culture. Based on these results, we herein propose, for the first time, a degradation pathway for amaranth. Our results suggest that Dec 1 degrades amaranth via the combined activities of peroxidase and hydrolase and reductase action.

  17. Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in Castellaniella defragrans

    PubMed Central

    2012-01-01

    Background Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of β-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid. A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events. Results Polar effects were absent in the in-frame deletion mutants C. defragrans Δldi and C. defragrans ΔgeoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype. Conclusions In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial

  18. The p38 mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis

    PubMed Central

    Schett, G; Zwerina, J; Firestein, G

    2009-01-01

    Chronic inflammatory processes are based on a sustained and tightly regulated communication network among different cells types. This network comprises extracellular mediators such as cytokines, chemokines and matrix-degrading proteases, which orchestrate the participation of cells in the chronic inflammatory process. The mirrors of this outside communication world are intracellular transcription factor pathways, which shuttle information about inflammatory stimuli to the cell nucleus. This review examines the function of one key signal transduction pathway of inflammation—the p38 mitogen-activated protein kinases (p38MAPK). The signalling pathway is considered as crucial for the induction and maintenance of chronic inflammation, and its components thus emerge as interesting molecular targets of small molecule inhibitors for controlling inflammation. This review not only summarises the current knowledge of activation, regulation and function of the p38MAPK pathway but also examines the role of this pathway in clinical disease. It gives an overview of current evidence of p38MAPK activation in inflammatory arthritis and elaborates the key molecular determinants which contribute to p38MAPK activation in joint disease. PMID:17827184

  19. Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase

    PubMed Central

    Nedvetsky, Pavel I.; Tabor, Vedrana; Tamma, Grazia; Beulshausen, Sven; Skroblin, Philipp; Kirschner, Aline; Mutig, Kerim; Boltzen, Mareike; Petrucci, Oscar; Vossenkämper, Anna; Wiesner, Burkhard; Bachmann, Sebastian; Rosenthal, Walter

    2010-01-01

    Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK–dependent pathway. PMID:20724536

  20. In situ protein-SIP highlights Burkholderiaceae as key players degrading toluene by para ring hydroxylation in a constructed wetland model.

    PubMed

    Lünsmann, Vanessa; Kappelmeyer, Uwe; Benndorf, René; Martinez-Lavanchy, Paula M; Taubert, Anja; Adrian, Lorenz; Duarte, Marcia; Pieper, Dietmar H; von Bergen, Martin; Müller, Jochen A; Heipieper, Hermann J; Jehmlich, Nico

    2016-04-01

    In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere, stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited. We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii. We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.

  1. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    SciTech Connect

    Mulari, Mika T.K. Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-05-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.

  2. A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast.

    PubMed

    Mathiassen, Søs G; Larsen, Ida B; Poulsen, Esben G; Madsen, Christian T; Papaleo, Elena; Lindorff-Larsen, Kresten; Kragelund, Birthe B; Nielsen, Michael L; Kriegenburg, Franziska; Hartmann-Petersen, Rasmus

    2015-08-21

    A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.

  3. Intranuclear protein transduction through a nucleoside salvage pathway.

    PubMed

    Hansen, James E; Tse, Chung-Ming; Chan, Grace; Heinze, Emil R; Nishimura, Robert N; Weisbart, Richard H

    2007-07-20

    Regulation of gene expression by intranuclear transduction of macromolecules such as transcription factors is an alternative to gene therapy for the treatment of numerous diseases. The identification of an effective intranuclear delivery vehicle and pathway for the transport of therapeutic macromolecules across plasma and nuclear membranes, however, has posed a significant challenge. The anti-DNA antibody fragment 3E10 Fv has received attention as a novel molecular delivery vehicle due to its penetration into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. Elucidation of the pathway that allows 3E10 Fv to cross cell membranes is critical to the development of new molecular therapies. Here we show that 3E10 Fv penetrates cells through a nucleoside salvage transporter. 3E10 Fv is unable to penetrate into cells deficient in the equilibrative nucleoside transporter ENT2, and reconstitution of ENT2 into ENT2-deficient cells restores 3E10 Fv transport into cell nuclei. Our results represent the first demonstration of protein transport through a nucleoside salvage pathway. We expect that our finding will facilitate a variety of methods of gene regulation in the treatment of human diseases, open up new avenues of research in nucleoside salvage pathways, and enhance our understanding of the pathophysiology of autoimmune diseases.

  4. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  5. Reduction-oxidation state and protein degradation in skeletal muscles of growing rats

    NASA Technical Reports Server (NTRS)

    Fagan, Julie M.; Tischler, Marc E.

    1986-01-01

    The relationship between the NAD redox state and protein degradation during growth was studied in isolated soleus and extensor digitorum longus muscles of 4- to 14-week-old rats. As muscle size increased with age, protein breakdown slowed and the muscles became progressively more reduced as shown by higher ratios of lactate/pyruvate in incubated and fresh-frozen muscle. Correlations were strong between redox state of protein degradation, and muscle mass, and between redox state and protein degradation. This relationship may be important in the slowing of muscle breakdown that occurs with age.

  6. UBE4B protein couples ubiquitination and sorting machineries to enable epidermal growth factor receptor (EGFR) degradation.

    PubMed

    Sirisaengtaksin, Natalie; Gireud, Monica; Yan, Qing; Kubota, Yoshihisa; Meza, Denisse; Waymire, Jack C; Zage, Peter E; Bean, Andrew J

    2014-01-31

    The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.

  7. Carbon and chlorine isotope analysis to identify abiotic degradation pathways of 1,1,1-trichloroethane.

    PubMed

    Palau, Jordi; Shouakar-Stash, Orfan; Hunkeler, Daniel

    2014-12-16

    This study investigates dual C-Cl isotope fractionation during 1,1,1-TCA transformation by heat-activated persulfate (PS), hydrolysis/dehydrohalogenation (HY/DH) and Fe(0). Compound-specific chlorine isotope analysis of 1,1,1-TCA was performed for the first time, and transformation-associated isotope fractionation ε bulk C and ε bulk Cl values were -4.0 ± 0.2‰ and no chlorine isotope fractionation with PS, -1.6 ± 0.2‰ and -4.7 ± 0.1‰ for HY/DH, -7.8 ± 0.4‰ and -5.2 ± 0.2‰ with Fe(0). Distinctly different dual isotope slopes (Δδ13C/Δδ37Cl): ∞ with PS, 0.33 ± 0.04 for HY/DH and 1.5 ± 0.1 with Fe(0) highlight the potential of this approach to identify abiotic degradation pathways of 1,1,1-TCA in the field. The trend observed with PS agreed with a C-H bond oxidation mechanism in the first reaction step. For HY/DH and Fe(0) pathways, different slopes were obtained although both pathways involve cleavage of a C-Cl bond in their initial reaction step. In contrast to the expected larger primary carbon isotope effects relative to chlorine for C-Cl bond cleavage, ε bulk C < ε bulk Cl was observed for HY/DH and in a similar range for reduction by Fe(0), suggesting the contribution of secondary chlorine isotope effects. Therefore, different magnitude of secondary chlorine isotope effects could at least be partly responsible for the distinct slopes between HY/DH and Fe(0) pathways. Following this dual isotope approach, abiotic transformation processes can unambiguously be identified and quantified.

  8. Type V Protein Secretion Pathway: the Autotransporter Story

    PubMed Central

    Henderson, Ian R.; Navarro-Garcia, Fernando; Desvaux, Mickaël; Fernandez, Rachel C.; Ala'Aldeen, Dlawer

    2004-01-01

    Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function. PMID:15590781

  9. Anaerobic Degradation of p-Ethylphenol by “Aromatoleum aromaticum” Strain EbN1: Pathway, Regulation, and Involved Proteins▿ †

    PubMed Central

    Wöhlbrand, Lars; Wilkes, Heinz; Halder, Thomas; Rabus, Ralf

    2008-01-01

    The denitrifying “Aromatoleum aromaticum” strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure (∼15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol- and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1. PMID:18539747

  10. The unfolded protein response (UPR) pathway in Cryptococcus

    PubMed Central

    Cheon, Seon Ah; Jung, Kwang-Woo; Bahn, Yong-Sun; Kang, Hyun Ah

    2014-01-01

    Unique and evolutionarily conserved signaling pathways allow an organism to sense, respond to, and adapt to internal and external environmental cues at its biological niche. In eukaryotic cells, the unfolded protein response (UPR) pathway regulates endoplasmic reticulum (ER) homeostasis upon exposure to environmental changes causing ER stress. The UPR pathway of Cryptococcus neoformans, an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals, consists of the evolutionarily conserved Ire1 kinase, a unique bZIP transcription factor, Hxl1, and the ER-resident molecular chaperone Kar2/BiP. Although the Cryptococcus UPR pathway regulates ER stress, antifungal drug resistance, and virulence in an Ire1/Hxl1-dependent manner, Ire1 has Hxl1-independent roles in capsule biosynthesis and thermotolerance. In this review, we highlight the conserved and unique features of the Cryptococcus UPR pathway compared with other fungal UPR systems and its importance in the pathogenesis of cryptococcosis and discuss future challenges in this field. PMID:24504058

  11. Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource

    PubMed Central

    Koike, Asako; Kobayashi, Yoshiyuki; Takagi, Toshihisa

    2003-01-01

    Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. PMID:12799355

  12. CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.

    PubMed

    Kashima, Lisa; Idogawa, Masashi; Mita, Hiroaki; Shitashige, Miki; Yamada, Tesshi; Ogi, Kazuhiro; Suzuki, Hiromu; Toyota, Minoru; Ariga, Hiroyoshi; Sasaki, Yasushi; Tokino, Takashi

    2012-04-13

    The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.

  13. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

    PubMed Central

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  14. Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4

    PubMed Central

    Shields, Malcolm S.; Montgomery, Stacy O.; Chapman, Peter J.; Cuskey, Stephen M.; Pritchard, P. H.

    1989-01-01

    o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of 18O- and 16O-labeled 3-methylcatechol from toluene in an atmosphere of 18O2 and 16O2 established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities. PMID:16347956

  15. Chemical degradation of proteins in the solid state with a focus on photochemical reactions.

    PubMed

    Mozziconacci, Olivier; Schöneich, Christian

    2015-10-01

    Protein pharmaceuticals comprise an increasing fraction of marketed products but the limited solution stability of proteins requires considerable research effort to prepare stable formulations. An alternative is solid formulation, as proteins in the solid state are thermodynamically less susceptible to degradation. Nevertheless, within the time of storage a large panel of kinetically controlled degradation reactions can occur such as, e.g., hydrolysis reactions, the formation of diketopiperazine, condensation and aggregation reactions. These mechanisms of degradation in protein solids are relatively well covered by the literature. Considerably less is known about oxidative and photochemical reactions of solid proteins. This review will provide an overview over photolytic and non-photolytic degradation reactions, and specially emphasize mechanistic details on how solid structure may affect the interaction of protein solids with light.

  16. Excretion pathways and ruminal disappearance of glyphosate and its degradation product aminomethylphosphonic acid in dairy cows.

    PubMed

    von Soosten, D; Meyer, U; Hüther, L; Dänicke, S; Lahrssen-Wiederholt, M; Schafft, H; Spolders, M; Breves, G

    2016-07-01

    From 6 balance experiments with total collection of feces and urine, samples were obtained to investigate the excretion pathways of glyphosate (GLY) in lactating dairy cows. Each experiment lasted for 26d. The first 21d served for adaptation to the diet, and during the remaining 5d collection of total feces and urine was conducted. Dry matter intake and milk yield were recorded daily and milk and feed samples were taken during the sampling periods. In 2 of the 6 experiments, at the sampling period for feces and urine, duodenal contents were collected for 5d. Cows were equipped with cannulas at the dorsal sac of the rumen and the proximal duodenum. Duodenal contents were collected every 2h over 5 consecutive days. The daily duodenal dry matter flow was measured by using chromium oxide as a volume marker. All samples (feed, feces, urine, milk and duodenal contents were analyzed for GLY and aminomethylphosphonic acid (AMPA). Overall, across the 6 experiments (n=32) the range of GLY intake was 0.08 to 6.67mg/d. The main proportion (61±11%; ±SD) of consumed GLY was excreted with feces; whereas excretion by urine was 8±3% of GLY intake. Elimination via milk was negligible. The GLY concentrations above the limit of quantification were not detected in any of the milk samples. A potential ruminal degradation of GLY to AMPA was derived from daily duodenal GLY flow. The apparent ruminal disappearance of GLY intake was 36 and 6%. In conclusion, the results of the present study indicate that the gastrointestinal absorption of GLY is of minor importance and fecal excretion represents the major excretion pathway. A degradation of GLY to AMPA by rumen microbes or a possible retention in the body has to be taken into account.

  17. Degradation pathways of lamotrigine under advanced treatment by direct UV photolysis, hydroxyl radicals, and ozone.

    PubMed

    Keen, Olya S; Ferrer, Imma; Michael Thurman, E; Linden, Karl G

    2014-12-01

    Lamotrigine is recently recognized as a persistent pharmaceutical in the water environment and wastewater effluents. Its degradation was studied under UV and ozone advanced oxidation treatments with reaction kinetics of lamotrigine with ozone (≈4 M(-1)s(-1)), hydroxyl radical [(2.1 ± 0.3) × 10(9)M(-1)s(-1)] and by UV photolysis with low and medium pressure mercury vapor lamps [quantum yields ≈0 and (2.7 ± 0.4)× 10(-4) respectively] determined. All constants were measured at pH 6 and at temperature ≈20°C. The results indicate that lamotrigine is slow to respond to direct photolysis or oxidation by ozone and no attenuation of the contaminant is expected in UV or ozone disinfection applications. The compound reacts rapidly with hydroxyl radicals indicating that advanced oxidation processes would be effective for its treatment. Degradation products were identified under each treatment process using accurate mass time-of-flight spectrometry and pathways of decay were proposed. The main transformation pathways in each process were: dechlorination of the benzene ring during direct photolysis; hydroxyl group addition to the benzene ring during the reaction with hydroxyl radicals; and triazine ring opening after reaction with ozone. Different products that form in each process may be to a varying degree less environmentally stable than the parent lamotrigine. In addition, a novel method of ozone quenching without addition of salts is presented. The new quenching method would allow subsequent mass spectrometry analysis without a solid phase extraction clean-up step. The method involves raising the pH of the sample to approximately 10 for a few seconds and lowering it back and is therefore limited to applications for which temporary pH change is not expected to affect the outcome of the analysis.

  18. Photocatalytic degradation of pesticide methomyl: determination of the reaction pathway and identification of intermediate products.

    PubMed

    Tamimi, M; Qourzal, S; Assabbane, A; Chovelon, J-M; Ferronato, C; Ait-Ichou, Y

    2006-05-01

    The degradation of pesticide methomyl in aqueous solution by UV-irradiation in the presence of TiO2 "Degussa P-25" has been studied. It was found that mineralisation to carbon dioxide, water, sulfate and ammonia took place during the process. The rate of photodecomposition of methomyl was measured using high performance liquid chromatography (HPLC), while its mineralization was followed using ion chromatography (IC), and total organic carbon (TOC) analysis. The identification of reaction intermediate products was carried out using coupled techniques HPLC-MS (electrospray ionization in positive mode) and a degradation pathway was proposed. Under our conditions, complete disappearance of 1.23 x 10(-4) mol l(-1) of pure pesticide occurred within 45 min of illumination and 80% TOC removal occurred in less than 4 h. Three main intermediates were identified resulting from (i) the rupture of the ester bond (or the N-O bond), (ii) the hydroxylation of methyl group borne by the nitrogen atom and (iii) the product resulting from the decarboxylation of the oxidized hydroxylated methyl group (photo-Kolbe reaction). In order to be sure that the photocatalytic results were consistent, hydrolysis and photolysis tests were performed. Photocatalysis proved to be an excellent new advanced oxidation technology (AOT) to eliminate methomyl present in water.

  19. Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences

    PubMed Central

    Boehm, Volker; Gerbracht, Jennifer V.; Marx, Marie-Charlotte; Gehring, Niels H.

    2016-01-01

    The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5′–3′ mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3′ UTRs of cytokine mRNAs reveals the contribution of 5′–3′ decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses. PMID:27917860

  20. Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9

    PubMed Central

    Martin, Vincent J. J.; Mohn, William W.

    2000-01-01

    We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. The dit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the α and β subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylE transcriptional fusion strains showed induction of ditA1 and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the dit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, a ditR::Kmr mutation in a ditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation. PMID:10850995

  1. Pathways for degrading TNT by Thu-Z: a Pantoea sp. strain.

    PubMed

    Zou, Liangdong; Lu, Diannan; Liu, Zheng

    2012-12-01

    2,4,6-Trinitrotoluene (TNT), an extensively used and versatile explosive, is harmful in soil and water. In the present study, four bacterial strains capable of degrading TNT have been isolated from contaminated sites and named as Thu-A, Thu-B, Thu-C, and Thu-Z. Thu-Z, which gave the highest degradation efficiency compared to the others, was assigned to the genus Pantoea according to its 16S rRNA gene. Similarities in both biochemical properties and morphology suggested that Thu-Z was a Pantoea sp. strain. Thu-Z was proved to be capable of using TNT as a sole nitrogen source by cleaving NO(2) from the nitroaromatic ring by direct aromatic ring reduction. Under nitrogen-limited conditions, 96.6 % N of TNT was consumed by Thu-Z for growth, which was determined in terms of NaNO(2). Trace nitro reduction metabolites such as 2,4-diamino-6-nitrotoluene (24Dam) and 2,6-diamino-4-nitrotoluene (26Dam) were identified in the presence of (NH(4))(2)SO(4). On the other hand, 4,4',6,6'-tetranitro-2,2'-azoxytoluene (22Azo) and 2,2',6,6'-tetranitro-4,4'-azoxytoluene (44Azo) were detected in the absence of (NH(4))(2)SO(4). These indicated the existence of a dual pathway for Thu-Z, while the direct aromatic ring reduction was predominant. Addition of a nitrogen source ((NH(4))(2)SO(4)) after inoculation stimulated the growth of Thu-Z and accelerated TNT degradation.

  2. Protein Kinases of the Hippo Pathway: Regulation and Substrates

    PubMed Central

    Avruch, Joseph; Zhou, Dawang; Fitamant, Julien; Bardeesy, Nabeel; Mou, Fan; Barrufet, Laura Regué

    2012-01-01

    The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP

  3. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation.

    PubMed

    Winter, Georg E; Buckley, Dennis L; Paulk, Joshiawa; Roberts, Justin M; Souza, Amanda; Dhe-Paganon, Sirano; Bradner, James E

    2015-06-19

    The development of effective pharmacological inhibitors of multidomain scaffold proteins, notably transcription factors, is a particularly challenging problem. In part, this is because many small-molecule antagonists disrupt the activity of only one domain in the target protein. We devised a chemical strategy that promotes ligand-dependent target protein degradation using as an example the transcriptional coactivator BRD4, a protein critical for cancer cell growth and survival. We appended a competitive antagonist of BET bromodomains to a phthalimide moiety to hijack the cereblon E3 ubiquitin ligase complex. The resultant compound, dBET1, induced highly selective cereblon-dependent BET protein degradation in vitro and in vivo and delayed leukemia progression in mice. A second series of probes resulted in selective degradation of the cytosolic protein FKBP12. This chemical strategy for controlling target protein stability may have implications for therapeutically targeting previously intractable proteins.

  4. The histamine degradative uptake pathway in human vascular endothelial cells and skin fibroblasts is dependent on extracellular Na+ and Cl-

    SciTech Connect

    Haddock, R.C.; Mack, P.; Leal, S.; Baenziger, N.L. )

    1990-08-25

    We have previously reported that human vascular endothelial cells and skin fibroblasts carry out degradation of (3H)histamine by a mechanism involving two successive enzymatic steps: imidazole ring tele-methylation by the cells' endogenous methyltransferase and subsequent amine oxidation by an exogenous diamine oxidase. Both histamine and the exogenous second enzyme in the pathway associate with the cells via separate binding sites or receptors. The enzymatic degradation process results in cellular accumulation of the proximal and distal metabolites tele-methylhistamine and 1-methyl-4-imidazoleacetic acid (MIAA). We have now demonstrated that this two-stage histamine degradative pathway is dependent on Na+ and Cl- in the extracellular environment. Accumulation of (3H) histamine-derived products is partially inhibited under conditions of Na+ deprivation and more substantially when Cl- is also withdrawn. The individual tele-methylation and amine oxidation enzymatic reactions themselves are unaffected or actually facilitated under these conditions. This indicates that it is the cellular mechanism for uptake coupled to the degradative pathway which reflects the cation and anion dependency. Restoration of degradative uptake displays a biphasic Na+ concentration curve, suggesting that the uptake process may be driven by multiple components. These findings indicate a role for both inward Na+ and Cl- ion movement in this cellular degradative uptake mechanism.

  5. Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway.

    PubMed

    Padrão, Juliana da Cruz; Cabral, Gabriel Rabello de Abreu; da Silva, Maria de Fátima Sarro; Seabra, Sergio Henrique; DaMatta, Renato Augusto

    2014-10-01

    Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.

  6. Text mining for metabolic pathways, signaling cascades, and protein networks.

    PubMed

    Hoffmann, Robert; Krallinger, Martin; Andres, Eduardo; Tamames, Javier; Blaschke, Christian; Valencia, Alfonso

    2005-05-10

    The complexity of the information stored in databases and publications on metabolic and signaling pathways, the high throughput of experimental data, and the growing number of publications make it imperative to provide systems to help the researcher navigate through these interrelated information resources. Text-mining methods have started to play a key role in the creation and maintenance of links between the information stored in biological databases and its original sources in the literature. These links will be extremely useful for database updating and curation, especially if a number of technical problems can be solved satisfactorily, including the identification of protein and gene names (entities in general) and the characterization of their types of interactions. The first generation of openly accessible text-mining systems, such as iHOP (Information Hyperlinked over Proteins), provides additional functions to facilitate the reconstruction of protein interaction networks, combine database and text information, and support the scientist in the formulation of novel hypotheses. The next challenge is the generation of comprehensive information regarding the general function of signaling pathways and protein interaction networks.

  7. In vitro degradation of the 32kDa PS II reaction centre protein

    SciTech Connect

    Eckenswiller, L.C.; Greenberg, B.M. )

    1989-04-01

    The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components.

  8. Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development[OPEN

    PubMed Central

    Nelson, Clark J.; Castleden, Ian

    2017-01-01

    We applied 15N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome. PMID:28138016

  9. Sequence and 3D structure based analysis of TNT degrading proteins in Arabidopsis thaliana.

    PubMed

    Bhattacherjee, Amrita; Mandal, Rahul Shubhra; Das, Santasabuj; Kundu, Sudip

    2014-03-01

    TNT, accidentally released at several manufacturing sites, contaminates ground water and soil. It has a toxic effect to algae and invertebrate, and chronic exposure to TNT also causes harmful effects to human. On the other hand, many plants including Arabidopsis thaliana have the ability to metabolize TNT either completely or at least to a reduced less toxic form. In A. thaliana, the enzyme UDP glucosyltransferase (UDPGT) can further conjugate the reduced forms 2-HADNT and 4-HADNT (2-hydroxylamino-4, 6- dinitrotoluene and 4-hydroxylamino-2, 6- dinitrotoluene) of TNT. Based on the experimental analysis, existing literature and phylogenetic analysis, it is evident that among 107 UDPGT proteins only six are involved in the TNT degrading process. A total of 13 UDPGT proteins including five of these TNT degrading proteins fall within the same group of phylogeny. Thus, these 13 UDPGT proteins have been classified into two groups, TNT-degrading and TNT-non-degrading proteins. To understand the differences in TNT-degrading capacities; using homology modeling we first predicted two structures, taking one representative sequence from both the groups. Next, we performed molecular docking of the modeled structure and TNT reduced form 2-hydroxylamino-4, 6- dinitrotoluene (2-HADNT). We observed that while the Trp residue located within the active site region of the TNT- degrading protein showed π-Cation interaction; such type of interaction was absent in TNT-non-degrading protein, as the respective Trp residue lay outside of the pocket in this case. We observed the conservation of this π-Cation interaction during MD simulation of TNT-degrading protein. Thus, the position and the orientation of the active site residue Trp could explain the presence and absence of TNT-degrading capacity of the UDPGT proteins.

  10. Forage Management Effects on Protein and Fiber Fractions, Protein Degradability, and Dry Matter Yield of Red Clover Conserved as Silage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to the action of o-quinones formed via polyphenol oxidase, conserved red clover (Trifolium pratense L.) contains abundant rumen undegradable protein (RUP), but inadequate rumen degradable protein (RDP) for dairy cattle. This study examined how forage management influences RDP, RUP, crude protein...

  11. Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway.

    PubMed

    Pettersson, Susanne; Sczaniecka, Matylda; McLaren, Lorna; Russell, Fiona; Gladstone, Karen; Hupp, Ted; Wallace, Maura

    2013-03-15

    The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.

  12. Silencing of SIAH1 in SH-SY5Y affects α-synuclein degradation pathway

    PubMed Central

    Xu, Jing; Zhang, Xin-Zhi; Zhang, Yong-Jin; Li, Xiu-Ming; Cai, Zeng-Lin; Li, Xiao-Min

    2015-01-01

    Seven in absentia homolog (SIAH) is a ubiquitin ligase that monoubiquitinates α-synuclein. Lewy bodies are characteristically rich in monoubiquitinated α-synuclein. We aimed to determine the effect of siRNA-SIAH1 on α-synuclein autophagy and UPS degradation in SH-SY5Y. SIAH1 expression was measured with real-time quantitative PCR and Western Blot. Cell proliferation was measured by CCK-8 assay; cell apoptosis assayed by flow cytometry. Relative protein expressions were measured by Western Blot. mRNA levels of relative protein were measured by real-time quantitative PCR. The expression of α-synuclein, LC3-II and SIAH1 were observed by confocal microscopy. We found: (1) Transfection efficiency of SIAH1-siRNA into SH-SY5 measured approximately 89% by flow cytometry. (2) siRNA silencing of SIAH1 promoted cellular proliferation and suppressed apoptosis. (3) Protein and mRNA expression of α-synuclein, LC3-II and p53 decreased after SIAH1 knockdown. E1 protein and mRNA levels increased after SIAH1 siRNA. These data show silencing SIAH1 increased cell proliferation and inhibited apoptosis in SH-SY5Y neuroblastoma cells. SIAH1 knockdown enhanced the clearance of non-aggregated α-synuclein by UPS. SIAH1 is a potential target for treatment of Parkinson’s disease. PMID:26722480

  13. Starvation-Dependent Regulation of Golgi Quality Control Links the TOR Signaling and Vacuolar Protein Sorting Pathways.

    PubMed

    Dobzinski, Niv; Chuartzman, Silvia G; Kama, Rachel; Schuldiner, Maya; Gerst, Jeffrey E

    2015-09-22

    Upon amino acid (AA) starvation and TOR inactivation, plasma-membrane-localized permeases rapidly undergo ubiquitination and internalization via the vacuolar protein sorting/multivesicular body (VPS-MVB) pathway and are degraded in the yeast vacuole. We now show that specific Golgi proteins are also directed to the vacuole under these conditions as part of a Golgi quality-control (GQC) process. The degradation of GQC substrates is dependent upon ubiquitination by the defective-for-SREBP-cleavage (DSC) complex, which was identified via genetic screening and includes the Tul1 E3 ligase. Using a model GQC substrate, GFP-tagged Yif1, we show that vacuolar targeting necessitates upregulation of the VPS pathway via proteasome-mediated degradation of the initial endosomal sorting complex required for transport, ESCRT-0, but not downstream ESCRT components. Thus, early cellular responses to starvation include the targeting of specific Golgi proteins for degradation, a phenomenon reminiscent of the inactivation of BTN1, the yeast Batten disease gene ortholog.

  14. Characterization of a novel β-cypermethrin-degrading Aspergillus niger YAT strain and the biochemical degradation pathway of β-cypermethrin.

    PubMed

    Deng, Weiqin; Lin, Derong; Yao, Kai; Yuan, Huaiyu; Wang, Zhilong; Li, Jianlong; Zou, Likou; Han, Xinfeng; Zhou, Kang; He, Li; Hu, Xinjie; Liu, Shuliang

    2015-10-01

    Aspergillus niger YAT strain was obtained from Chinese brick tea (Collection number: CGMCC 10,568) and identified on the basis of morphological characteristics and internal transcribed spacer (ITS) sequence. The strain could degrade 54.83 % of β-cypermethrin (β-CY; 50 mg L(-1)) in 7 days and 100 % of 3-phenoxybenzoic acid (3-PBA; 100 mg L(-1)) in 22 h. The half-lives of β-CY and 3-PBA range from 3.573 to 11.748 days and from 5.635 to 12.160 h, respectively. The degradation of β-CY and 3-PBA was further described using first-order kinetic models. The pathway and mechanism of β-CY degraded by YAT were investigated by analyzing the degraded metabolites through high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Relevant enzymatic activities and substrate utilization were also investigated. β-CY degradation products were analyzed. Results indicated that YAT strain transformed β-CY into 3-PBA. 3-PBA was then gradually transformed into permethric acid, protocatechuic acid, 3-hydroxy-5-phenoxy benzoic acid, gallic acid, and phenol gradually. The YAT strain can also effectively degrade these metabolites. The results indicated that YAT strain has potential applications in bioremediation of pyrethroid insecticide (PI)-contaminated environments and fermented food.

  15. The roles of unfolded protein response pathways in Chlamydia pathogenesis.

    PubMed

    George, Zenas; Omosun, Yusuf; Azenabor, Anthony A; Partin, James; Joseph, Kahaliah; Ellerson, Debra; He, Qing; Eko, Francis; Bandea, Claudiu; Svoboda, Pavel; Pohl, Jan; Black, Carolyn M; Igietseme, Joseph U

    2016-12-08

    Chlamydia is an obligate intracellular bacterium that relies on host cell for essential nutrients and ATP for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the three UPR pathway transducers: Protein kinase RNA-activated (PKR)-like ER kinase (PERK), Inositol-requiring enzyme-1α (IRE1α), and Activating transcription factor-6α (ATF6α) were activated during Chlamydia infection. The kinase activity of PERK and RNase of IRE1α mediated the upregulation of Hexokinase II and production of ATP via substrate level phosphorylation. In addition, the activation of PERK and IRE1α promoted autophagy formation and apoptosis resistance or host survival. Moreover, the activation of IRE1α resulted in the generation of spliced XBP1 and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia.

  16. How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway.

    PubMed

    Dyson, H Jane; Wright, Peter E

    2017-01-17

    Although each type of protein fold and in some cases individual proteins within a fold classification can have very different mechanisms of folding, the underlying biophysical and biochemical principles that operate to cause a linear polypeptide chain to fold into a globular structure must be the same. In an aqueous solution, the protein takes up the thermodynamically most stable structure, but the pathway along which the polypeptide proceeds in order to reach that structure is a function of the amino acid sequence, which must be the final determining factor, not only in shaping the final folded structure, but in dictating the folding pathway. A number of groups have focused on a single protein or group of proteins, to determine in detail the factors that influence the rate and mechanism of folding in a defined system, with the hope that hypothesis-driven experiments can elucidate the underlying principles governing the folding process. Our research group has focused on the folding of the globin family of proteins, and in particular on the monomeric protein apomyoglobin. Apomyoglobin (apoMb) folds relatively slowly (∼2 s) via an ensemble of obligatory intermediates that form rapidly after the initiation of folding. The folding pathway can be dissected using rapid-mixing techniques, which can probe processes in the millisecond time range. Stopped-flow measurements detected by circular dichroism (CD) or fluorescence spectroscopy give information on the rates of folding events. Quench-flow experiments utilize the differential rates of hydrogen-deuterium exchange of amide protons protected in parts of the structure that are folded early; protection of amides can be detected by mass spectrometry or proton nuclear magnetic resonance spectroscopy (NMR). In addition, apoMb forms an intermediate at equilibrium at pH ∼ 4, which is sufficiently stable for it to be structurally characterized by solution methods such as CD, fluorescence and NMR spectroscopies, and the

  17. Identification of G protein-coupled receptor signaling pathway proteins in marine diatoms using comparative genomics

    PubMed Central

    2013-01-01

    Background The G protein-coupled receptor (GPCR) signaling pathway plays an essential role in signal transmission and response to external stimuli in mammalian cells. Protein components of this pathway have been characterized in plants and simpler eukaryotes such as yeast, but their presence and role in unicellular photosynthetic eukaryotes have not been determined. We use a comparative genomics approach using whole genome sequences and gene expression libraries of four diatoms (Pseudo-nitzschia multiseries, Thalassiosira pseudonana, Phaeodactylum tricornutum and Fragilariopsis cylindrus) to search for evidence of GPCR signaling pathway proteins that share sequence conservation to known GPCR pathway proteins. Results The majority of the core components of GPCR signaling were well conserved in all four diatoms, with protein sequence similarity to GPCRs, human G protein α- and β-subunits and downstream effectors. There was evidence for the Gγ-subunit and thus a full heterotrimeric G protein only in T. pseudonana. Phylogenetic analysis of putative diatom GPCRs indicated similarity but deep divergence to the class C GPCRs, with branches basal to the GABAB receptor subfamily. The extracellular and intracellular regions of these putative diatom GPCR sequences exhibited large variation in sequence length, and seven of these sequences contained the necessary ligand binding domain for class C GPCR activation. Transcriptional data indicated that a number of the putative GPCR sequences are expressed in diatoms under various stress conditions in culture, and that many of the GPCR-activated signaling proteins, including the G protein, are also expressed. Conclusions The presence of sequences in all four diatoms that code for the proteins required for a functional mammalian GPCR pathway highlights the highly conserved nature of this pathway and suggests a complex signaling machinery related to environmental perception and response in these unicellular organisms. The lack of

  18. A Critical Appraisal of Quantitative Studies of Protein Degradation in the Framework of Cellular Proteostasis

    PubMed Central

    Alvarez-Castelao, Beatriz; Ruiz-Rivas, Carmen; Castaño, José G.

    2012-01-01

    Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish “definitive evidence” for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover. PMID:23119163

  19. Degradation behavior of soy protein-wheat gluten films in simulated soil conditions.

    PubMed

    Park, S K; Hettiarachchy, N S; Were, L

    2000-07-01

    Films containing soy protein and wheat gluten were exposed to simulated farmland soil mix over a period of 30 days and monitored for degradation. The simulated farmland soil mix (topsoil/sand/Sunshine compost/vermiculite, 59:6:25:10, wt %) was mixed and stored at ambient humidity (48-55%) and temperature (20-24 degrees C); the soil mix was constantly maintained at 15% moisture by weight. Research focused on evaluating the effectiveness of gluten and cysteine additions on biodegradable behavior in the simulated farmland soil conditions. The four types of films, soy protein (S:G 1:0); soy protein with cysteine addition (S:G 1:0 + CYS); soy protein-wheat gluten (S:G 4:1); and soy protein-wheat gluten with cysteine addition (S:G 4:1 + CYS), were prepared at pH 7. 0 for degradation studies. Soy protein-gluten film rapidly degraded with 50% weight loss in about 10 days and with up to 95% weight loss in 30 days. Tensile strength and elongation of all soy protein-gluten films significantly decreased in 3 days. However, cysteine addition delayed the degradation rate of soy protein-gluten films. Soy protein-wheat gluten film disintegrated after 20 days in the simulated farmland soil environment. These results suggest that wheat gluten and cysteine addition to soy protein-based films could delay degradation rates due to their high disulfide contents.

  20. Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

    PubMed Central

    Labunskyy, Vyacheslav M.; Gerashchenko, Maxim V.; Delaney, Joe R.; Kaya, Alaattin; Kennedy, Brian K.; Kaeberlein, Matt; Gladyshev, Vadim N.

    2014-01-01

    Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity. PMID:24391512

  1. Proteomic analysis of changes in the Kandelia candel chloroplast proteins reveals pathways associated with salt tolerance.

    PubMed

    Wang, Lingxia; Pan, Dezhuo; Li, Jian; Tan, Fanglin; Hoffmann-Benning, Susanne; Liang, Wenyu; Chen, Wei

    2015-02-01

    The plant chloroplast is one of the most sensitive organelles in response to salt stress. Chloroplast proteins extracted from seedling leaves were separated by two-dimensional gel electrophoresis (2-DE). More than 600 protein spots could be distinguished on each gel. Fifty-eight differentially expressed protein spots were detected, of which 46 could be identified through matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). These proteins were found to be involved in multiple aspects of chloroplast metabolism pathways such as photosynthesis, ATP synthesis, detoxification and antioxidation processes, nitrogen assimilation and fixation, protein metabolism, and tetrapyrrole biosynthesis. The results indicated that K. candel could withstand up to 500 mM NaCl stress for a measured period of 3 days, by maintaining normal or high photosynthetic electron transfer efficiency and an only slightly stimulated Calvin cycle. Meanwhile, we found that ROS scavenging, nitrogen assimilation, protein degradation and chaperone function in chloroplasts were also of importance for salt tolerance of K. candel. The ultrastructural and physiological data agree with chloroplast proteome results. These findings allow further exploration of our knowledge on salt adaptation in woody halophytes and may contribute to the development of more salt-tolerant plants in the future.

  2. The ESCRT-III-interacting deubiquitinating enzyme AMSH3 is essential for degradation of ubiquitinated membrane proteins in Arabidopsis thaliana.

    PubMed

    Katsiarimpa, Anthi; Muñoz, Alfonso; Kalinowska, Kamila; Uemura, Tomohiro; Rojo, Enrique; Isono, Erika

    2014-04-01

    Post-translational modification by ubiquitin plays a key role in the regulation of endocytic degradation in which ubiquitinated plasma membrane cargos are transported to the vacuole for degradation dependent on the ESCRT (endosomal sorting complex required for transport) machinery. Arabidopsis AMSH3 (ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 3) is a deubiquitinating enzyme that interacts with at least two subunits of the ESCRT-III machinery, VPS2.1 and VPS24.1. amsh3 null mutation causes seedling lethality, and amsh3 null mutants show defects in multiple intracellular trafficking pathways. In this study, we further analyzed the amsh3 mutant phenotype and showed that amsh3 accumulates membrane-associated ubiquitinated proteins, supporting the indication that AMSH3 functions in ubiquitin-mediated endocytic degradation. In accordance with this, an enzymatic inactive variant of AMSH3 inhibits the AvrPtoB-dependent endocytic degradation of CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1). Furthermore, we showed that the interaction of AMSH3 with ESCRT-III is important for its function in planta. Together, our data indicate the importance of AMSH3 and the AMSH3-ESCRT-III interaction for deubiquitination and degradation of ubiquitinated membrane substrates in plants.

  3. RNA Binding Proteins in the miRNA Pathway.

    PubMed

    Connerty, Patrick; Ahadi, Alireza; Hutvagner, Gyorgy

    2015-12-26

    microRNAs (miRNAs) are short ~22 nucleotides (nt) ribonucleic acids which post-transcriptionally regulate gene expression. miRNAs are key regulators of all cellular processes, and the correct expression of miRNAs in an organism is crucial for proper development and cellular function. As a result, the miRNA biogenesis pathway is highly regulated. In this review, we outline the basic steps of miRNA biogenesis and miRNA mediated gene regulation focusing on the role of RNA binding proteins (RBPs). We also describe multiple mechanisms that regulate the canonical miRNA pathway, which depends on a wide range of RBPs. Moreover, we hypothesise that the interaction between miRNA regulation and RBPs is potentially more widespread based on the analysis of available high-throughput datasets.

  4. Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

    PubMed

    Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

    2017-03-01

    Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein.

  5. Isoprenylation of RhoB is necessary for its degradation. A novel determinant in the complex regulation of RhoB expression by the mevalonate pathway.

    PubMed

    Stamatakis, Konstantinos; Cernuda-Morollón, Eva; Hernández-Perera, Octavio; Pérez-Sala, Dolores

    2002-12-20

    Statins improve vascular functions by mechanisms independent from their cholesterol-lowering effect. Rho GTPases are emerging as key targets for the vascular effects of statins. RhoB is a short-lived, early-response inducible protein involved in receptor endocytosis, apoptosis, and gene expression. Here we show that statins regulate RhoB expression by acting at multiple levels. Simvastatin increased RhoB protein levels by 8- to 10-fold. This effect was related to a depletion of isoprenoid intermediates, as deduced from the observation that several metabolites of the cholesterol biosynthetic pathway, namely, mevalonate and geranylgeranyl-pyrophosphate, attenuated simvastatin-induced RhoB up-regulation. Moreover, prenyltransferase inhibitors mimicked simvastatin effect. Cholesterol supplementation did not prevent simvastatin-elicited up-regulation but increased RhoB levels per se. Simvastatin moderately augmented RhoB transcript levels, but markedly impaired the degradation of RhoB protein, which accumulated in the cytosol in its non-isoprenylated form. Inhibition of RhoB isoprenylation was apparently required for simvastatin-induced up-regulation, because levels of an isoprenylation-deficient RhoB mutant were not affected by simvastatin. Moreover, this mutant was found to be markedly more stable than the wild-type protein. These results show that RhoB isoprenylation is necessary for rapid turnover of this protein and identify a novel link between the cholesterol biosynthetic pathway and the regulation of G-protein expression.

  6. Complete genome sequence of the anaerobic, protein-degrading hyperthermophilic crenarchaeon Desulfurococcus kamchatkensis.

    PubMed

    Ravin, Nikolai V; Mardanov, Andrey V; Beletsky, Alexey V; Kublanov, Ilya V; Kolganova, Tatiana V; Lebedinsky, Alexander V; Chernyh, Nikolai A; Bonch-Osmolovskaya, Elizaveta A; Skryabin, Konstantin G

    2009-04-01

    Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extrachromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis. The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS607 and IS200/IS605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis. Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organism's nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.

  7. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals.

    PubMed

    Karabinova, Pavla; Kubelka, Michal; Susor, Andrej

    2011-10-01

    Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.

  8. Revisiting Heat-Damaged Protein and Ruminal Degradation Kinetics in Heated Hays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies utilizing conventional (45-kg) hay bales have shown that acid-detergent insoluble CP (ADICP), ruminal CP degradation rate (Kd), and rumen degradable protein (RDP) are related to various measures of spontaneous heating during bale storage in simple linear relationships that frequentl...

  9. Enhanced cytokinin synthesis in tobacco plants expressing PSARK::IPT prevents the degradation of photosynthetic protein complexes during drought.

    PubMed

    Rivero, Rosa M; Gimeno, Jacinta; Van Deynze, Allen; Walia, Harkamal; Blumwald, Eduardo

    2010-11-01

    To identify genes associated with the cytokinin-induced enhanced drought tolerance, we analyzed the transcriptome of wild-type and transgenic tobacco (Nicotiana tabacum 'SR1') plants expressing P(SARK)::IPT (for senescence-associated receptor kinase::isopentenyltransferase) grown under well-watered and prolonged water deficit conditions using the tomato GeneChip. During water deficit, the expression of genes encoding components of the carotenoid pathway leading to ABA biosynthesis was enhanced in the wild-type plants, but repressed in the transgenic plants. On the other hand, transgenic plants displayed higher transcript abundance of genes involved in the brassinosteroid biosynthetic pathways. Several genes coding for proteins associated with Chl synthesis, light reactions, the Calvin-Benson cycle and photorespiration were induced in the transgenic plants. Notably, increased transcript abundance of genes associated with PSII, the cytochrome b(6)/f complex, PSI, NADH oxidoreductase and the ATP complex was found in the P(SARK)::IPT plants. The increased transcript abundance was assessed by quantitative PCR and the increased protein levels were confirmed by Western blots. Our results indicated that while the photosynthetic apparatus in the wild-type plants was degraded, photosynthesis in the transgenic plants was not affected and photosynthetic proteins were not degraded. During water deficit, wild-type plants displayed a significant reduction in electron transfer and photochemical quenching, with a marked increase in non-photochemical quenching, suggesting a decrease in energy transfer to the PSII core complexes and an increase in cyclic electron transfer reactions.

  10. Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

    PubMed Central

    Choung-Hee Lee, Elaine; Deonarine, Andrew; Strange, Kevin

    2012-01-01

    Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage

  11. Glucocorticoids alleviate intestinal ER stress by enhancing protein folding and degradation of misfolded proteins.

    PubMed

    Das, Indrajit; Png, Chin Wen; Oancea, Iulia; Hasnain, Sumaira Z; Lourie, Rohan; Proctor, Martina; Eri, Rajaraman D; Sheng, Yong; Crane, Denis I; Florin, Timothy H; McGuckin, Michael A

    2013-06-03

    Endoplasmic reticulum (ER) stress in intestinal secretory cells has been linked with colitis in mice and inflammatory bowel disease (IBD). Endogenous intestinal glucocorticoids are important for homeostasis and glucocorticoid drugs are efficacious in IBD. In Winnie mice with intestinal ER stress caused by misfolding of the Muc2 mucin, the glucocorticoid dexamethasone (DEX) suppressed ER stress and activation of the unfolded protein response (UPR), substantially restoring goblet cell Muc2 production. In mice lacking inflammation, a glucocorticoid receptor antagonist increased ER stress, and DEX suppressed ER stress induced by the N-glycosylation inhibitor, tunicamycin (Tm). In cultured human intestinal secretory cells, in a glucocorticoid receptor-dependent manner, DEX suppressed ER stress and UPR activation induced by blocking N-glycosylation, reducing ER Ca(2+) or depleting glucose. DEX up-regulated genes encoding chaperones and elements of ER-associated degradation (ERAD), including EDEM1. Silencing EDEM1 partially inhibited DEX's suppression of misfolding-induced ER stress, showing that DEX enhances ERAD. DEX inhibited Tm-induced MUC2 precursor accumulation, promoted production of mature mucin, and restored ER exit and secretion of Winnie mutant recombinant Muc2 domains, consistent with enhanced protein folding. In IBD, glucocorticoids are likely to ameliorate ER stress by promoting correct folding of secreted proteins and enhancing removal of misfolded proteins from the ER.

  12. Degradation of nicosulfuron by a novel isolated bacterial strain Klebsiella sp. Y1: condition optimization, kinetics and degradation pathway.

    PubMed

    Wang, Lin; Zhang, Xiaolin; Li, Yongmei

    2016-01-01

    A novel bacterial strain Klebsiella sp. Y1 was isolated from the soil of a constructed wetland, and it was identified based on the 16S rDNA sequence analysis. The co-metabolic degradation of nicosulfuron with glucose by Klebsiella sp. Y1 was investigated. The response surface methodology analysis indicated that the optimal pH and temperature were 7.0 and 35 °C, respectively, for the degradation of nicosulfuron. Under the optimal conditions, the degradation of nicosulfuron fitted Haldane kinetics model well. The removal of nicosulfuron was triggered by the acidification of glucose, which accelerated the hydrolysis of nicosulfuron. Then, the C-N bond of the sulfonylurea bridge was attacked and cleaved. Finally, the detected intermediate 2-amino-4,6-dimethoxypyrimidine was further biodegraded.

  13. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444 issue 3)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-14

    Highlights: • The 2000–2634 aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  14. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444, isse 4)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-21

    Highlights: • The 2000–2634aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  15. Connecting Lignin-Degradation Pathway with Pre-Treatment Inhibitor Sensitivity of Cupriavidus necator

    SciTech Connect

    Wang, W.; Yang, S.; Hunsinger, G. B.; Pienkos, P. T.; Johnson, D. K.

    2014-05-27

    In order to produce lignocellulosic biofuels economically, the complete release of monomers from the plant cell wall components, cellulose, hemicellulose, and lignin, through pre-treatment and hydrolysis (both enzymatic and chemical), and the efficient utilization of these monomers as carbon sources, is crucial. In addition, the identification and development of robust microbial biofuel production strains that can tolerate the toxic compounds generated during pre-treatment and hydrolysis is also essential. In this work, Cupriavidus necator was selected due to its capabilities for utilizing lignin monomers and producing polyhydroxylbutyrate (PHB), a bioplastic as well as an advanced biofuel intermediate. We characterized the growth kinetics of C. necator in pre-treated corn stover slurry as well as individually in the pre-sence of 11 potentially toxic compounds in the saccharified slurry. We found that C. necator was sensitive to the saccharified slurry produced from dilute acid pre-treated corn stover. Five out of 11 compounds within the slurry were characterized as toxic to C. necator, namely ammonium acetate, furfural, hydroxymethylfurfural (HMF), benzoic acid, and p-coumaric acid. Aldehydes (e.g., furfural and HMF) were more toxic than the acetate and the lignin degradation products benzoic acid and p-coumaric acid; furfural was identified as the most toxic compound. Although toxic to C. necator at high concentration, ammonium acetate, benzoic acid, and p-coumaric acid could be utilized by C. necator with a stimulating effect on C. necator growth. Consequently, the lignin degradation pathway of C. necator was reconstructed based on genomic information and literature. The efficient conversion of intermediate catechol to downstream products of cis,cis-muconate or 2-hydroxymuconate-6-semialdehyde may help improve the robustness of C. necator to benzoic acid and p-coumaric acid as well as improve PHB productivity.

  16. The Whole Genome Sequence of Sphingobium chlorophenolicum L-1: Insights into the Evolution of the Pentachlorophenol Degradation Pathway

    SciTech Connect

    Copley, Shelley D.; Rokicki, Joseph; Turner, Pernilla; Daligault, Hajnalka E.; Nolan, Matt; Land, Miriam L

    2012-01-01

    Sphingobium chlorophenolicum Strain L-1 can mineralize the toxic pesticide pentachlorophenol (PCP). We have sequenced the genome of S. chlorophenolicum Strain L-1. The genome consists of a primary chromosome that encodes most of the genes for core processes, a secondary chromosome that encodes primarily genes that appear to be involved in environmental adaptation, and a small plasmid. The genes responsible for degradation of PCP are found on chromosome 2. We have compared the genomes of S. chlorophenolicum Strain L-1 and Sphingobium japonicum, a closely related Sphingomonad that degrades lindane. Our analysis suggests that the genes encoding the first three enzymes in the PCP degradation pathway were acquired via two different horizontal gene transfer events, and the genes encoding the final two enzymes in the pathway were acquired from the most recent common ancestor of these two bacteria.

  17. Insights from 14C into C loss pathways in degraded peatlands

    NASA Astrophysics Data System (ADS)

    Evans, Martin; Evans, Chris; Allott, Tim; Stimson, Andrew; Goulsbra, Claire

    2016-04-01

    Peatlands are important global stores of terrestrial carbon. Lowered water tables due to changing climate and direct or indirect human intervention produce a deeper aerobic zone and have the potential to enhance loss of stored carbon from the peat profile. The quasi continuous accumulation of organic matter in active peatlands means that the age of fluvial dissolved organic carbon exported from peatland systems is related to the source depth in the peat profile. Consequently 14C analysis of DOC in waters draining peatlands has the potential not only to tell us about the source of fluvial carbon and the stability of the peatland but also about the dominant hydrological pathways in the peatland system. This paper will present new radiocarbon determinations from peatland streams draining the heavily eroded peatlands of the southern Pennine uplands in the UK. These blanket peatland systems are highly degraded, with extensive bare peat and gully erosion resulting from air pollution during the industrial revolution, overgrazing, wildfire and climatic changes. Deep and extensive gullying has significantly modified the hydrology of these systems leading to local and more widespread drawdown of water table. 14C data from DOC in drainage waters are presented from two catchments; one with extensive gully erosion and the other with a combination of gully erosion and sheet erosion of the peat. At the gully eroded site DOC in drainage waters is as old as 160 BP but at the site with extensive sheet erosion dates of up to 1069 BP are amongst the oldest recorded from blanket peatland globally These data indicate significant degradation of stored carbon from the eroding peatlands. Initial comparisons of the 14C data with modelled water table for the catchments and depth-age curves for catchment peats suggests that erosion of the peat surface, allowing decomposition of exposed older organic material is a potential mechanism producing aged carbon from the eroded catchment. This

  18. BIM(EL), an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation.

    PubMed

    Wiggins, Ceri M; Tsvetkov, Peter; Johnson, Mark; Joyce, Claire L; Lamb, Christopher A; Bryant, Nia J; Komander, David; Shaul, Yosef; Cook, Simon J

    2011-03-15

    BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

  19. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast.

    PubMed

    Maurer, Matthew J; Spear, Eric D; Yu, Allen T; Lee, Evan J; Shahzad, Saba; Michaelis, Susan

    2016-07-07

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones.

  20. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

    PubMed Central

    Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  1. Pathway logic modeling of protein functional domains in signal transduction.

    PubMed

    Talcott, C; Eker, S; Knapp, M; Lincoln, P; Laderoute, K

    2004-01-01

    Protein functional domains (PFDs) are consensus sequences within signaling molecules that recognize and assemble other signaling components into complexes. Here we describe the application of an approach called Pathway Logic to the symbolic modeling signal transduction networks at the level of PFDs. These models are developed using Maude, a symbolic language founded on rewriting logic. Models can be queried (analyzed) using the execution, search and model-checking tools of Maude. We show how signal transduction processes can be modeled using Maude at very different levels of abstraction involving either an overall state of a protein or its PFDs and their interactions. The key insight for the latter is our algebraic representation of binding interactions as a graph.

  2. Serum protein adsorption and excretion pathways of metal nanoparticles

    PubMed Central

    Vinluan, Rodrigo D; Zheng, Jie

    2015-01-01

    While the synthesis of metal nanoparticles (NPs) with fascinating optical and electronic properties have progressed dramatically and their potential biomedical applications were also well demonstrated in the past decade, translation of metal NPs into the clinical practice still remains a challenge due to their severe accumulation in the body. Herein, we give a brief review on size-dependent material properties of metal NPs and their potential biomedical applications, followed by a summary of how structural parameters such as size, shape and charge influence their interactions with serum protein adsorption, cellular uptake and excretion pathways. Finally, the future challenges in minimizing serum protein adsorption and expediting clinical translation of metal NPs were also discussed. PMID:26377047

  3. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  4. Putrescine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells.

    PubMed

    Kong, Xiangfeng; Wang, Xiaoqiu; Yin, Yulong; Li, Xilong; Gao, Haijun; Bazer, Fuller W; Wu, Guoyao

    2014-11-01

    Insufficient placental growth is a major factor contributing to intrauterine growth retardation in mammals. There is growing evidence that putrescine produced from arginine (Arg) and proline via ornithine decarboxylase is a key regulator of angiogenesis, embryogenesis, as well as placental and fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that putrescine stimulates protein synthesis by activating the mechanistic target of rapamycin (mTOR) signaling pathway in porcine trophectoderm cell line 2 cells. The cells were cultured for 2 to 4 days in customized Arg-free Dulbecco modified Eagle Ham medium containing 0, 10, 25, or 50 μM putrescine or 100 μM Arg. Cell proliferation, protein synthesis, and degradation, as well as the abundance of total and phosphorylated mTOR, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. Our results indicate that putrescine promotes cell proliferation and protein synthesis in a dose- and time-dependent manner, which was inhibited by difluoro-methylornithine (an inhibitor of ornithine decarboxylase). Moreover, supplementation of culture medium with putrescine increased the abundance of phosphorylated mTOR and its downstream targets, 4EBP1 and p70 S6K1 proteins. Collectively, these findings reveal a novel and important role for putrescine in regulating the mTOR signaling pathway in porcine placental cells. We suggest that dietary supplementation with or intravenous administration of putrescine may provide a new and effective strategy to improve survival and growth of embryos/fetuses in mammals.

  5. Monitoring of the Enzymatic Degradation of Protein Corona and Evaluating the Accompanying Cytotoxicity of Nanoparticles.

    PubMed

    Ma, Zhifang; Bai, Jing; Jiang, Xiue

    2015-08-19

    Established nanobio interactions face the challenge that the formation of nanoparticle-protein corona complexes shields the inherent properties of the nanoparticles and alters the manner of the interactions between nanoparticles and biological systems. Therefore, many studies have focused on protein corona-mediated nanoparticle binding, internalization, and intracellular transportation. However, there are a few studies to pay attention to if the corona encounters degradation after internalization and how the degradation of the protein corona affects cytotoxicity. To fill this gap, we prepared three types of off/on complexes based on gold nanoparticles (Au NPs) and dye-labeled serum proteins and studied the extracellular and intracellular proteolytic processes of protein coronas as well as their accompanying effects on cytotoxicity through multiple evaluation mechanisms, including cell viability, adenosine triphosphate (ATP) content, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS). The proteolytic process was confirmed by recovery of the fluorescence of the dye-labeled protein molecules that was initially quenched by Au NPs. Our results indicate that the degradation rate of protein corona is dependent on the type of the protein based on systematical evaluation of the extracellular and intracellular degradation processes of the protein coronas formed by human serum albumin (HSA), γ-globulin (HGG), and serum fibrinogen (HSF). Degradation is the fastest for HSA corona and the slowest for HSF corona. Notably, we also find that the Au NP-HSA corona complex induces lower cell viability, slower ATP production, lower MMP, and higher ROS levels. The cytotoxicity of the nanoparticle-protein corona complex may be associated with the protein corona degradation process. All of these results will enrich the database of cytotoxicity induced by nanomaterial-protein corona complexes.

  6. Novel organization of catechol meta pathway genes in the nitrobenzene degrader Comamonas sp. JS765 and its evolutionary implication.

    PubMed

    He, Zhongqi; Parales, Rebecca E; Spain, Jim C; Johnson, Glenn R

    2007-02-01

    The catechol meta cleavage pathway is one of the central metabolic pathways for the degradation of aromatic compounds. A novel organization of the pathway genes, different from that of classical soil microorganisms, has been observed in Sphingomonas sp HV3 and Pseudomonas sp. DJ77. In a Comamonas sp. JS765, cdoE encoding catechol 2,3-dioxygenase shares a common ancestry only with tdnC of a Pseudomonas putida strain, while codG encoding 2-hydroxymuconic semialdehyde dehydrogenase shows a higher degree of similarity to those genes in classical bacteria. Located between cdoE and cdoG are several putative genes, whose functions are unknown. These genes are not found in meta pathway operons of other microorganisms with the exception of cdoX2, which is similar to cmpX in strain HV3. Therefore, the gene cluster in JS765 reveals a third type of gene organization of the meta pathway.

  7. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    PubMed

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

  8. Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

    PubMed

    Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

    2015-05-13

    Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

  9. Effects of gamma irradiation on chemical composition and ruminal protein degradation of canola meal

    NASA Astrophysics Data System (ADS)

    Shawrang, P.; Nikkhah, A.; Zare-Shahneh, A.; Sadeghi, A. A.; Raisali, G.; Moradi-Shahrebabak, M.

    2008-07-01

    Gamma irradiation of canola meal (at doses of 25, 50 and 75 kGy) could alter its ruminal protein degradation characteristics by cross-linking of the polypeptide chains. This processing resulted in decrease (linear effect, P<0.001) of ruminal protein degradation and increase (linear effect, P<0.001) of intestinal protein digestibility. The results showed that gamma irradiation at doses higher than 25 kGy can be used as a cross-linking agent to improve protein properties of supplements in ruminant nutrition.

  10. The twin arginine protein transport pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies.

    PubMed

    Joshi, Madhumita V; Mann, Stefan G; Antelmann, Haike; Widdick, David A; Fyans, Joanna K; Chandra, Govind; Hutchings, Matthew I; Toth, Ian; Hecker, Michael; Loria, Rosemary; Palmer, Tracy

    2010-07-01

    Summary Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and DeltatatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The DeltatatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.

  11. Autopalmitoylation of TEAD Proteins Regulates Transcriptional Output of Hippo Pathway

    PubMed Central

    Chan, PuiYee; Han, Xiao; Zheng, Baohui; DeRan, Michael; Yu, Jianzhong; Jarugumilli, Gopala K.; Deng, Hua; Pan, Duojia; Luo, Xuelian; Wu, Xu

    2016-01-01

    TEA domain (TEAD) transcription factors bind to the co-activator YAP/TAZ, and regulate the transcriptional output of Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches fatty acid (palmitate) to cysteine residues, and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities, and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs, and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation is required for TEAD’s binding to YAP/TAZ, but dispensable for the binding to Vgll4 tumor suppressor. In addition, palmitoylation does not alter TEAD’s localization. Moreover, TEAD palmitoylation-deficient mutants impaired TAZ-mediated muscle differentiation in vitro, and Yorkie-mediated tissue overgrowth in Drosophila in vivo. Our study directly linked autopalmitoylation to the transcriptional regulation of Hippo pathway. PMID:26900866

  12. Protein export through the bacterial flagellar type III export pathway.

    PubMed

    Minamino, Tohru

    2014-08-01

    For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

  13. ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation.

    PubMed

    Seo, Ji Hae; Park, Ji-Hyeon; Lee, Eun Ji; Vo, Tam Thuy Lu; Choi, Hoon; Kim, Jun Yong; Jang, Jae Kyung; Wee, Hee-Jun; Lee, Hye Shin; Jang, Se Hwan; Park, Zee Yong; Jeong, Jaeho; Lee, Kong-Joo; Seok, Seung-Hyeon; Park, Jin Young; Lee, Bong Jin; Lee, Mi-Ni; Oh, Goo Taeg; Kim, Kyu-Won

    2016-10-06

    Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.

  14. ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation

    PubMed Central

    Seo, Ji Hae; Park, Ji-Hyeon; Lee, Eun Ji; Vo, Tam Thuy Lu; Choi, Hoon; Kim, Jun Yong; Jang, Jae Kyung; Wee, Hee-Jun; Lee, Hye Shin; Jang, Se Hwan; Park, Zee Yong; Jeong, Jaeho; Lee, Kong-Joo; Seok, Seung-Hyeon; Park, Jin Young; Lee, Bong Jin; Lee, Mi-Ni; Oh, Goo Taeg; Kim, Kyu-Won

    2016-01-01

    Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70