The ubiquitin/proteasome pathway from Lemna minor subjected to heat shock

Exposure of Lemna minor L. to high temperatures leads to an initial decrease in the ubiquitin (Ub) monomer pool size and the accumulation of high molecular mass Ub-protein conjugates, possibly reflecting an increment in the supply of protein substrates to the Ub/proteasome pathway. Alternative explanations include, for example, changes in the transcription rates of one or more pathway components. To measure the effect of heat shock on the simultaneous rates of transcription of selected genes encoding five Ub pathway components (Ub, E1, E2, β subunit and ATPase subunit of the 26S proteasome), a semi-quantitative RT-PCR method was developed using cDNA normalized against the housekeeping gene encoding the 18S ribosomal RNA. Whilst Ub transcription is abruptly increased, there is a moderate increment in the transcription of E1 and the β subunit, a moderate reduction in the transcription of the ATPase subunit and a marked reduction in the case of E2, indicating a differential transcription pattern of the various components of the Ub/proteasome pathway in L. minor subjected to high temperatures. These observations suggest that the increment in the Ub/proteasome pathway intermediates is due to an augmented supply of substrates derived from the stress-induced damage imposed on the cellular proteins. The initial build up of intermediates occurs not only at the expense of the preexisting pool of free Ub, but also as a result of the prompt increase in Ub expressio

Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2

<p>Abstract</p> <p>Background</p> <p>Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The <it>Mycobacterium tuberculosis </it>(MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium.</p> <p>Results</p> <p>We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in <it>Escherichia coli</it>. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides.</p> <p>We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers.</p> <p>Conclusion</p> <p>Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes.</p

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Peer reviewedPublisher PD

Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker.

DnaK/Hsp70 chaperones form oligomers of poorly understood structure and functional significance. Site-specific proteolysis and crosslinking were used to probe the architecture of oligomers formed by the endoplasmic reticulum (ER) Hsp70, BiP. These were found to consist of adjacent protomers engaging the interdomain linker of one molecule in the substrate binding site of another, attenuating the chaperone function of oligomeric BiP. Native gel electrophoresis revealed a rapidly-modulated reciprocal relationship between the burden of unfolded proteins and BiP oligomers and slower equilibration between oligomers and inactive, covalently-modified BiP. Lumenal ER calcium depletion caused rapid oligomerization of mammalian BiP and a coincidental diminution in substrate binding, pointing to the relative inertness of the oligomers. Thus, equilibration between inactive oligomers and active monomeric BiP is poised to buffer fluctuations in ER unfolded protein load on a rapid timescale attainable neither by inter-conversion of active and covalently-modified BiP nor by the conventional unfolded protein response.Supported by grants from the Wellcome Trust (Wellcome 084812/Z/08/Z) the European Commission (EU FP7 Beta-Bat No: 277713), a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (Wellcome 100140) and a US Public Health Service grant NIH-GM54068 (to LH). DR is a Wellcome Trust Principal Research Fellow.This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.0896

Thioredoxin fold as homodimerization module in the putative chaperone ERp29 : NMR structures of the domains and experimental model of the 51 kDa dimer

Funding Information: We thank Dr. William J. Griffiths for mass spectrometric analysis; Dr. Andrei Kaikkonen for the calculation of paramagnetic relaxation enhancements; and the Swedish NMR Center for access to the 800 MHz NMR spectrometer. This work was supported by the Swedish Natural Science Research Council, Swedish Medical Research Council, Swedish Society for Medical Research, and by an FRN grant for the 500 MHz NMR spectrometer. M.B. received a scholarship from the Swedish Institute and the Royal Swedish Academy. Copyright: Copyright 2012 Elsevier B.V., All rights reserved.Background: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. Results: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. Conclusions: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.publishersversionPeer reviewe

Comparative Proteomic Analysis of Methanothermobacter themautotrophicus ΔH in Pure Culture and in Co-Culture with a Butyrate-Oxidizing Bacterium

To understand the physiological basis of methanogenic archaea living on interspecies H2 transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H2 supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F420-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed

Genome-wide single-cell-level screen for protein abundance and localization changes in response to DNA damage in S. cerevisiae

An effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed. Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF. We conclude that cellular responses can navigate different routes according to the extent of damage, relying on both expression and localization changes of specific proteins.National Cancer Institute (U.S.) (R01-CA055042 (now NIEHS R01-ES022872))Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant NIEHS P30-ES002109)National Cancer Institute (U.S.) (KI Center Grant U54-CA112967)National Cancer Institute (U.S.) (Cancer Center Support Grant P30-CA14051)National Institute of Environmental Health Sciences (R01-ES022872)MIT Faculty Start-up FundMassachusetts Institute of Technology. Computational and Systems Biology Initiative (Merck & Co. Postdoctoral Fellowship

Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradatio

Genome-Wide Fitness and Expression Profiling Implicate Mga2 in Adaptation to Hydrogen Peroxide

Caloric restriction extends lifespan, an effect once thought to involve attenuation of reactive oxygen species (ROS) generated by aerobic metabolism. However, recent evidence suggests that caloric restriction may in fact raise ROS levels, which in turn provides protection from acute doses of oxidant through a process called adaptation. To shed light on the molecular mechanisms of adaptation, we designed a series of genome-wide deletion fitness and mRNA expression screens to identify genes involved in adaptation to hydrogen peroxide. Combined with known transcriptional interactions, the integrated data implicate Yap1 and Skn7 as central transcription factors of both the adaptive and acute oxidative responses. They also identify the transcription factors Mga2 and Rox1 as active exclusively in the adaptive response and show that Mga2 is essential for adaptation. These findings are striking because Mga2 and Rox1 have been thought to control the response to hypoxic, not oxidative, conditions. Expression profiling of mga2Δ and rox1Δ knockouts shows that these factors most strongly regulate targets in ergosterol, fatty-acid, and zinc metabolic pathways. Direct quantitation of ergosterol reveals that its basal concentration indeed depends on Mga2, but that Mga2 is not required for the decrease in ergosterol observed during adaptation

Stability of Metabolic Correlations under Changing Environmental Conditions in Escherichia coli – A Systems Approach

Background: Biological systems adapt to changing environments by reorganizing their cellular and physiological program with metabolites representing one important response level. Different stresses lead to both conserved and specific responses on the metabolite level which should be reflected in the underlying metabolic network. Methodology/Principal Findings: Starting from experimental data obtained by a GC-MS based high-throughput metabolic profiling technology we here develop an approach that: (1) extracts network representations from metabolic condition-dependent data by using pairwise correlations, (2) determines the sets of stable and condition-dependent correlations based on a combination of statistical significance and homogeneity tests, and (3) can identify metabolites related to the stress response, which goes beyond simple observations about the changes of metabolic concentrations. The approach was tested with Escherichia coli as a model organism observed under four different environmental stress conditions (cold stress, heat stress, oxidative stress, lactose diauxie) and control unperturbed conditions. By constructing the stable network component, which displays a scale free topology and small-world characteristics, we demonstrated that: (1) metabolite hubs in this reconstructed correlation networks are significantly enriched for those contained in biochemical networks such as EcoCyc, (2) particular components of the stable network are enriched for functionally related biochemical pathways, and (3) independently of the response scale, based on their importance in the reorganization of the correlation network a set of metabolites can be identified which represent hypothetical candidates for adjusting to a stress-specific response. Conclusions/Significance: Network-based tools allowed the identification of stress-dependent and general metabolic correlation networks. This correlation-network-based approach does not rely on major changes in concentration to identify metabolites important for stress adaptation, but rather on the changes in network properties with respect to metabolites. This should represent a useful complementary technique in addition to more classical approaches