Loading...

Follow Portland Press - Biochemical Journal on Feedspot

Continue with Google
Continue with Facebook
or

Valid

Bacterial capsules have evolved to be at the forefront of the cell envelope, making them an essential element of bacterial biology. Efforts to understand the Mycobacterium tuberculosis (Mtb) capsule began more than 60 years ago, but the relatively recent development of mycobacterial genetics combined with improved chemical and immunological tools have revealed a more refined view of capsule molecular composition. A glycogen-like α-glucan is the major constituent of the capsule, with lower amounts of arabinomannan and mannan, proteins and lipids. The major Mtb capsular components mediate interactions with phagocytes that favor bacterial survival. Vaccination approaches targeting the mycobacterial capsule have proven successful in controlling bacterial replication. Although the Mtb capsule is composed of polysaccharides of relatively low complexity, the concept of antigenic variability associated with this structure has been suggested by some studies. Understanding how Mtb shapes its envelope during its life cycle is key to developing anti-infective strategies targeting this structure at the host–pathogen interface.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

Antioxidant systems modulate oxidant-based signaling networks and excessive removal of oxidants can prevent beneficial acclimation responses. Evidence from mutant, transgenic, and locally adapted natural plant systems is used to interpret differences in the capacity for antioxidation and formulate hypotheses for future inquiry. We focus on the first line of chloroplast antioxidant defense, pre-emptive thermal dissipation of excess absorbed light (monitored as nonphotochemical fluorescence quenching, NPQ) as well as on tocopherol-based antioxidation. Findings from NPQ-deficient and tocopherol-deficient mutants that exhibited enhanced biomass production and/or enhanced foliar water-transport capacity are reviewed and discussed in the context of the impact of lower levels of antioxidation on plant performance in hot/dry conditions, under cool temperature, and in the presence of biotic stress. The complexity of cellular redox-signaling networks is related to the complexity of environmental and endogenous inputs as well as to the need for intensified training and collaboration in the study of plant–environment interactions across biological sub-disciplines.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

Saccharomyces cerevisiae Spp1, a plant homeodomain (PHD) finger containing protein, is a critical subunit of the histone H3K4 methyltransferase complex of proteins associated with Set1 (COMPASS). The chromatin binding affinity of the PHD finger of Spp1 has been proposed to modulate COMPASS activity. During meiosis, Spp1 plays another role in promoting programmed double-strand break (DSB) formation by binding H3K4me3 via its PHD finger and interacting with a DSB protein, Mer2. However, how the Spp1 PHD finger performs site-specific readout of H3K4me3 is still not fully understood. In the present study, we determined the crystal structure of the highly conserved Spp1 N-terminal domain (Sc_Spp1NTD) in complex with the H3K4me3 peptide. The structure shows that Sc_Spp1NTD comprises a PHD finger responsible for methylated H3K4 recognition and a C3H-type zinc finger necessary to ensure the overall structural stability. Our isothermal titration calorimetry results show that binding of H3K4me3 to Sc_Spp1NTD is mildly inhibited by H3R2 methylation, weakened by H3T6 phosphorylation, and abrogated by H3T3 phosphorylation. This histone modification cross-talk, which is conserved in the Saccharomyces pombe and mammalian orthologs of Sc_Spp1 in vitro, can be rationalized structurally and might contribute to the roles of Spp1 in COMPASS activity regulation and meiotic recombination.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

Recently, Guenter Schwarz and colleagues published an elegant study in the Biochemical Journal (2019) 476, 1805–1815 which combines kinetic and spectroscopic studies with protein engineering to provide a mechanism for sulfite oxidase (SO)-catalyzed nitrite reduction that yields nitric oxide (NO). This work is noteworthy as it demonstrates that (i) for NO generation, both sulfite and nitrite must bind to the same molybdenum (Mo) center; (ii) upon sulfite reduction, Mo is reduced from +6 (MoVI) to +4 (MoIV) and MoIV reduces nitrite to NO yielding MoV; (iii) the heme moiety, linked to the Mo-center by an 11 amino acid residue tether, gets reduced by intramolecular electron transfer (IET) resulting in MoV being oxidized to MoVI; (iv) the reduced heme transfers its electron to a second nitrite molecule converting it to NO; (v) the authors demonstrate steady-state NO production in the presence of the natural electron acceptor cytochrome c; (vi) Finally, the authors use protein engineering to shorten the heme tether to reduce the heme-Mo-center distance with the aim of increasing NO production. Consequently, the rate of IET to cytochrome c is decreased but the enzymatic turnover rate for NO production is increased by ~10-fold. This paper is unique as it provides strong evidence for a novel mechanism for steady-state NO production for human mitochondrial SO and serves as a potential template for studying NO production mechanisms in other enzymes by integrating the information gained from enzyme kinetics with EPR and UV/vis spectroscopy and protein engineering.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid β-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

Metabolic remodeling plays an essential role in the pathophysiology of heart failure (HF). Many studies have shown that the disruption of phosphoinositide-dependent protein kinase-1 (PDK1) caused severe and lethal HF; however, the metabolic pattern of PDK1 deletion remains ambiguous. 1H nuclear magnetic resonance-based metabolomics was applied to explore the altered metabolic pattern in Pdk1-deficient mice. Principle component analysis showed significant separation as early as 4 weeks of age, and dysfunction of metabolism precedes a morphological change in Pdk1-deficient mice. A time trajectory plot indicated that disturbed metabolic patterns were related to the pathological process of the HF in Pdk1-deficient mice, rather than the age of mice. Metabolic profiles demonstrated significantly increased levels of acetate, glutamate, glutamine, and O-phosphocholine in Pdk1 deletion mice. Levels of lactate, alanine, glycine, taurine, choline, fumarate, IMP, AMP, and ATP were significantly decreased compared with controls. Furthermore, PDK1 knockdown decreased the oxygen consumption rate in H9C2 cells as determined using a Seahorse XF96 Analyzer. These findings imply that the disruption of metabolism and impaired mitochondrial activity might be involved in the pathogenesis of HF with PDK1 deletion.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 
Portland Press - Biochemical Journal by Chapman, K. D., Aziz, M., Dyer, J. .. - 1w ago

Lipid droplets (LDs) are organelles that compartmentalize nonbilayer-forming lipids in the aqueous cytoplasm of cells. They are ubiquitous in most organisms, including in animals, protists, plants and microorganisms. In eukaryotes, LDs are believed to be derived by a budding and scission process from the surface of the endoplasmic reticulum, and this occurs concomitantly with the accumulation of neutral lipids, most often triacylglycerols and steryl esters. Overall, the mechanisms underlying LD biogenesis are difficult to generalize, in part because of the involvement of different sets of both evolutionarily conserved and organism-specific LD-packaging proteins. Here, we briefly compare and contrast these proteins and the allied processes responsible for LD biogenesis in cells of animals, yeasts and plants.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

The mitogen-activated protein kinase ERK1/2 (ERKs, extracellular-regulated protein kinases) plays important roles in a wide spectrum of cellular processes and have been implicated in many disease states. The spatiotemporal regulation of ERK activity has been extensively studied. However, scarce information has been available regarding the quality control of the kinases to scavenge malfunctioning ERKs. Using site-specific mutagenesis and mass spectrometry, we found that the disruption of the conserved H-bond between Y210 and E237 of ERK1 through point mutation at or naturally occurring nitration on Y210 initiates a quality control program dependent on chaperon systems and CHIP (C-terminal of Hsp70-interacting protein)-mediated ubiquitination and degradation. The H-bond is also important for the quality control of ERK2, but through a distinct mechanism. These findings clearly demonstrate how malfunctioning ERKs are eliminated when cells are in certain stress conditions or unhealthy states, and could represent a general mechanism for scavenging malfunctioning kinases in stress conditions.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2–-dependent insertion of a Mg2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (Kd 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD–H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL–MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.

  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

Calcium signaling is essential for embryonic development but the signals upstream of calcium are only partially understood. Here, we investigate the role of the intracellular glutathione redox potential in calcium signaling using the Chac1 protein of zebrafish. A member of the -glutamylcyclotransferase family of enzymes, the zebrafish Chac1 is a glutathione-degrading enzyme that acts only on reduced glutathione. The zebrafish chac1 expression was seen early in development, and in the latter stages, in the developing muscles, brain and heart. The chac1 knockdown was embryonic lethal, and the developmental defects were seen primarily in the myotome, brain and heart where chac1 was maximally expressed. The phenotypes could be rescued by the WT Chac1 but not by the catalytically inactive Chac1 that was incapable of degrading glutathione. The ability of chac1 to alter the intracellular glutathione redox potential in the live animals was examined using Grx1-roGFP2. The chac1 morphants lacked the increased degree of cellular oxidation seen in the WT zebrafish. As calcium is also known to be critical for the developing myotomes, brain and heart, we further investigated if the chac1 knockdown phenotypes were a consequence of the lack of calcium signals. We observed using GCaMP6s, that calcium transients normally seen in the developing embryos were strongly attenuated in these knockdowns. The study thus identifies Chac1 and the consequent change in intracellular glutathione redox potential as important upstream activators of calcium signaling during development.

Read for later

Articles marked as Favorite are saved for later viewing.
close
  • Show original
  • .
  • Share
  • .
  • Favorite
  • .
  • Email
  • .
  • Add Tags 

Separate tags by commas
To access this feature, please upgrade your account.
Start your free month
Free Preview