scholarly journals Clp-Dependent Proteolysis Down-Regulates Central Metabolic Pathways in Glucose-Starved Bacillus subtilis

2007 ◽  
Vol 190 (1) ◽  
pp. 321-331 ◽  
Author(s):  
Ulf Gerth ◽  
Holger Kock ◽  
Ilja Kusters ◽  
Stephan Michalik ◽  
Robert L. Switzer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Pathways ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nucleotide Metabolism ◽  
Clp Proteases ◽  
In The Wild ◽  
Branched Chain ◽  
Expression Program ◽  
Intracellular Proteolysis

ABSTRACT Entry into stationary phase in Bacillus subtilis is linked not only to a redirection of the gene expression program but also to posttranslational events such as protein degradation. Using 35S-labeled methionine pulse-chase labeling and two-dimensional polyacrylamide gel electrophoresis we monitored the intracellular proteolysis pattern during glucose starvation. Approximately 200 protein spots diminished in the wild-type cells during an 8-h time course. The degradation rate of at least 80 proteins was significantly reduced in clpP, clpC, and clpX mutant strains. Enzymes of amino acid and nucleotide metabolism were overrepresented among these Clp substrate candidates. Notably, several first-committed-step enzymes for biosynthesis of aromatic and branched-chain amino acids, cell wall precursors, purines, and pyrimidines appeared as putative Clp substrates. Radioimmunoprecipitation demonstrated GlmS, IlvB, PurF, and PyrB to be novel ClpCP targets. Our data imply that Clp proteases down-regulate central metabolic pathways upon entry into a nongrowing state and thus contribute to the adaptation to nutrient starvation. Proteins that are obviously nonfunctional, unprotected, or even “unemployed” seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited.

scholarly journals Role of CcpA in Regulation of the Central Pathways of Carbon Catabolism in Bacillus subtilis

1999 ◽  
Vol 181 (22) ◽  
pp. 6996-7004 ◽  
Author(s):  
Steffen Tobisch ◽  
Daniela Zühlke ◽  
Jörg Bernhardt ◽  
Jörg Stülke ◽  
Michael Hecker
Keyword(s):  
Bacillus Subtilis ◽  
Glucose Repression ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tca Cycle ◽  
Transcriptional Analysis ◽  
Overflow Metabolism ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
In The Wild ◽  
Almost All

ABSTRACT The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi,pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpAmutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by theccpA mutation. Our data suggest (i) that ccpAmutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.

scholarly journals Itaconate Alters Succinate and Coenzyme A Metabolism via Inhibition of Mitochondrial Complex II and Methylmalonyl-CoA Mutase

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 117
Author(s):  
Thekla Cordes ◽  
Christian M. Metallo
Keyword(s):  
Amino Acid ◽  
Metabolic Pathways ◽  
Mammalian Cells ◽  
Coenzyme A ◽  
Cultured Cells ◽  
Tca Cycle ◽  
Regulatory Function ◽  
Reversible Inhibitor ◽  
Complex Ii ◽  
Branched Chain

Itaconate is a small molecule metabolite that is endogenously produced by cis-aconitate decarboxylase-1 (ACOD1) in mammalian cells and influences numerous cellular processes. The metabolic consequences of itaconate in cells are diverse and contribute to its regulatory function. Here, we have applied isotope tracing and mass spectrometry approaches to explore how itaconate impacts various metabolic pathways in cultured cells. Itaconate is a competitive and reversible inhibitor of Complex II/succinate dehydrogenase (SDH) that alters tricarboxylic acid (TCA) cycle metabolism leading to succinate accumulation. Upon activation with coenzyme A (CoA), itaconyl-CoA inhibits adenosylcobalamin-mediated methylmalonyl-CoA (MUT) activity and, thus, indirectly impacts branched-chain amino acid (BCAA) metabolism and fatty acid diversity. Itaconate, therefore, alters the balance of CoA species in mitochondria through its impacts on TCA, amino acid, vitamin B12, and CoA metabolism. Our results highlight the diverse metabolic pathways regulated by itaconate and provide a roadmap to link these metabolites to potential downstream biological functions.

CLEAVAGE PATHWAY,AND SPECIFICITY OF LEUCOCYTE ELASTASE AS COMPARED TO PLASMIN DURING FIBRINOGEN DEGRADATION

1987 ◽  
Author(s):  
L Goretzki ◽  
E Miller ◽  
A Henschen
Keyword(s):  
Amino Acid ◽  
Time Course ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Reversed Phase ◽  
Cleavage Sites ◽  
Human Fibrinogen ◽  
Leucocyte Elastase ◽  
N Terminus

Plasmin and leucocyte elastase are regarded as the two medically most important fibrin(ogen)-degrading proteolytic enzymes. There is, however, a considerable difference in information available about the cleavage specificities and fragmentation pathways of these two enzymes. Degradation by plasmin has been studied already for a long time in great detail so that now the time course of the degradation, the cleavage sites and the functional properties of many fragments are well known. In contrast, relatively little is known about the degradation by leucocyte elastase, except that the overall cleavage pattern resembles that obtained with plasminIn this investigation the leucocyte elastase-mediated degradation of fibrinogen has been examined by means of proteinchemi-cal methods. Human fibrinogen was incubated with human enzyme material for various periods of time and at some different enzyme concentrations. The split products formed at the various stages were isolated in pure form by gel filtration followed by reversed-phase high-performance liquid chromatography. The fragments were identified by N-terminal amino acid sequence and amino acid composition. The course of the degradation was also monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis. All cleavage patterns were compared with the corresponding patterns from plasmic degradation. It could be confirmed that X-, D- and E-like fragments are formed also with elastase. However, several early elastolytic Aα-chain fragments are characteristically different from plasmic fragments. The previously identified N-terminal cleavage site in the Aα-chain, i.e. after position 21, was found to be the most important site in this region of fibrinogen. The very early degradation of the Aα-chain N-terminus by elastase is in strong contrast to the stability against plasmin. Several cleavage sites in N-terminal region of the Bβ-chain were observed, though the low amino acid specificity of elastase partly hampered the identification. The γ-chain N-terminus was found to be as highly stable towards elastase as towards plasmin. The results are expected to contribute to the understanding of the role of leucocyte elastase in pathophysiologic fibrino(geno)lysis

scholarly journals Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

2008 ◽  
Vol 190 (17) ◽  
pp. 5841-5854 ◽  
Author(s):  
Helen Ting ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Metabolic Pathways ◽  
Synthetic Lethality ◽  
Nucleotide Metabolism ◽  
Genetic Interactions ◽  
Single Defect ◽  
Damage Repair ◽  
Mutant Combination ◽  
Dna Incorporation

ABSTRACT Synthetic lethality is inviability of a double-mutant combination of two fully viable single mutants, commonly interpreted as redundancy at an essential metabolic step. The dut-1 defect in Escherichia coli inactivates dUTPase, causing increased uracil incorporation in DNA and known synthetic lethalities [SL(dut) mutations]. According to the redundancy logic, most of these SL(dut) mutations should affect nucleotide metabolism. After a systematic search for SL(dut) mutants, we did identify a single defect in the DNA precursor metabolism, inactivating thymidine kinase (tdk), that confirmed the redundancy explanation of synthetic lethality. However, we found that the bulk of mutations interacting genetically with dut are in DNA repair, revealing layers of damage of increasing complexity that uracil-DNA incorporation sends through the chromosomal metabolism. Thus, we isolated mutants in functions involved in (i) uracil-DNA excision (ung, polA, and xthA); (ii) double-strand DNA break repair (recA, recBC, and ruvABC); and (iii) chromosomal-dimer resolution (xerC, xerD, and ftsK). These mutants in various DNA repair transactions cannot be redundant with dUTPase and instead reveal “defect-damage-repair” cycles linking unrelated metabolic pathways. In addition, two SL(dut) inserts (phoU and degP) identify functions that could act to support the weakened activity of the Dut-1 mutant enzyme, suggesting the “compensation” explanation for this synthetic lethality. We conclude that genetic interactions with dut can be explained by redundancy, by defect-damage-repair cycles, or as compensation.

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

scholarly journals Metabolic aspects of the secretion of stored compounds from blood platelets. The effect of NaF at different pH on nucleotide metabolism and function of washed platelets

1981 ◽  
Vol 194 (1) ◽  
pp. 187-192 ◽  
Author(s):  
E H Mürer ◽  
K Davenport ◽  
E Siojo ◽  
H J Day
Keyword(s):  
Time Course ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Fluoride Concentration ◽  
Indirect Evidence ◽  
Inhibitory Effect ◽  
Nucleotide Metabolism ◽  
Wide Range ◽  
And Function ◽  
Special Circumstances

The purpose of this study was to investigate the response of human blood platelets to fluoride at different pH. The results were as follows. (1) Fluoride induced secretion faster and at a lower concentration when pH was lowered. (2) Platelets exposed to 2 mM-fluoride at 0 degrees C at pH 5.3 underwent secretion when first pH and then temperature was raised, although no secretion was seen at 2 mM-fluoride concentration in the absence of the preincubation at low pH. (3) The concentration of [14C]ATP in platelets decreased steeply in response to fluoride before induction of secretion. Addition of antimycin blocked or partly inhibited secretion. Fluoride thus exerts an inhibitory effect on platelet glycolysis before induction of secretion. (4) Fluoride accumulated in the platelet pellet by a time course that preceded secretion. The accumulation was faster and greater at pH 6 than at 7.4. These four points are taken as indirect evidence that fluoride has to penetrate to the interior of the platelet to induce secretion. The activation takes place over a wide range of acid pH in contrast with induction of platelet function via the outside of the plasma membrane. In addition evidence is presented that the salvage pathway may under special circumstances play an important role in the re-synthesis of platelet adenine nucleotides.

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