scholarly journals The interactome of 2-Cys peroxiredoxins in Plasmodium falciparum

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Christina Brandstaedter ◽  
Claire Delahunty ◽  
Susanne Schipper ◽  
Stefan Rahlfs ◽  
John R. Yates ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Active Site ◽  
Cellular Protein ◽  
Mitochondrial Proteins ◽  
Target Proteins ◽  
Cellular Processes ◽  
Mixed Disulfide ◽  
Functional Analyses ◽  
Cell Proteome ◽  
Active Site Mutants

Abstract Peroxiredoxins (Prxs) are crucially involved in maintaining intracellular H2O2 homeostasis via their peroxidase activity. However, more recently, this class of proteins was found to also transmit oxidizing equivalents to selected downstream proteins, which suggests an important function of Prxs in the regulation of cellular protein redox relays. Using a pull-down assay based on mixed disulfide fishing, we characterized the thiol-dependent interactome of cytosolic Prx1a and mitochondrial Prx1m from the apicomplexan malaria parasite Plasmodium falciparum (Pf). Here, 127 cytosolic and 20 mitochondrial proteins that are components of essential cellular processes were found to interact with PfPrx1a and PfPrx1m, respectively. Notably, our data obtained with active-site mutants suggests that reducing equivalents might also be transferred from Prxs to target proteins. Initial functional analyses indicated that the interaction with Prx can strongly impact the activity of target proteins. The results provide initial insights into the interactome of Prxs at the level of a eukaryotic whole cell proteome. Furthermore, they contribute to our understanding of redox regulatory principles and thiol-dependent redox relays of Prxs in subcellular compartments.

scholarly journals Structural basis for UFM1 transfer from UBA5 to UFC1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar ◽  
Prasanth Padala ◽  
Jamal Fahoum ◽  
Fouad Hassouna ◽  
Tomer Tsaban ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structural Basis ◽  
Adenylation Domain ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Processes ◽  
Enzyme Cascade ◽  
Linear Sequence ◽  
C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

scholarly journals Thioredoxin A Active-Site Mutants Form Mixed Disulfide Dimers That Resemble Enzyme–Substrate Reaction Intermediates

2008 ◽  
Vol 379 (3) ◽  
pp. 520-534 ◽  
Author(s):  
Thijs R.H.M. Kouwen ◽  
Juni Andréll ◽  
Rianne Schrijver ◽  
Jean-Yves F. Dubois ◽  
Megan J. Maher ◽  
...  
Keyword(s):  
Active Site ◽  
Reaction Intermediates ◽  
Mixed Disulfide ◽  
Enzyme Substrate ◽  
Active Site Mutants

scholarly journals Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yi Zhang ◽  
Kyle Brown ◽  
Yucong Yu ◽  
Ziad Ibrahim ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cell Nucleus ◽  
Active Site ◽  
Histone Acetyltransferase ◽  
Histone H3 ◽  
Catalytic Core ◽  
Cellular Processes ◽  
Storage Pool ◽  
Core Components ◽  
Acetyltransferase P300

AbstractThe transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

scholarly journals Oligoribonuclease is the primary degradative enzyme for pGpG inPseudomonas aeruginosathat is required for cyclic-di-GMP turnover

2015 ◽  
Vol 112 (36) ◽  
pp. E5048-E5057 ◽  
Author(s):  
Mona W. Orr ◽  
Gregory P. Donaldson ◽  
Geoffrey B. Severin ◽  
Jingxin Wang ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Half Life ◽  
Biofilm Formation ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Capillary Action ◽  
Diguanylate Cyclases ◽  
Active Site Mutants ◽  
Degradative Enzyme

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A fromPseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆ornstrains ofP. aeruginosaPA14 for pGpG stability. The lysates from ∆ornshowed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆ornstrain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulatedpelpromoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆ornstrain in apel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆ornstrain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

Kinetic and Structural Analysis of Active Site Mutants of Monofunctional NAD-Dependent 5,10-Methylenetetrahydrofolate Dehydrogenase fromSaccharomyces cerevisiae†

Biochemistry ◽  
2005 ◽  
Vol 44 (39) ◽  
pp. 13163-13171 ◽  
Author(s):  
Wendi Wagner ◽  
Andrew P. Breksa ◽  
Arthur F. Monzingo ◽  
Dean R. Appling ◽  
Jon D. Robertus
Keyword(s):  
Structural Analysis ◽  
Active Site ◽  
Methylenetetrahydrofolate Dehydrogenase ◽  
Active Site Mutants

scholarly journals Akt2 Regulates Cardiac Metabolism and Cardiomyocyte Survival

2006 ◽  
Vol 281 (43) ◽  
pp. 32841-32851 ◽  
Author(s):  
Brian DeBosch ◽  
Nandakumar Sambandam ◽  
Carla Weinheimer ◽  
Michael Courtois ◽  
Anthony J. Muslin
Keyword(s):  
Pressure Overload ◽  
Cardiac Metabolism ◽  
Cellular Protein ◽  
Growth Responses ◽  
Infarct Zone ◽  
Targeted Disruption ◽  
Cellular Processes ◽  
Ligand Stimulation ◽  
And Function

The Akt family of serine-threonine kinases participates in diverse cellular processes, including the promotion of cell survival, glucose metabolism, and cellular protein synthesis. All three known Akt family members, Akt1, Akt2 and Akt3, are expressed in the myocardium, although Akt1 and Akt2 are most abundant. Previous studies demonstrated that Akt1 and Akt3 overexpression results in enhanced myocardial size and function. Yet, little is known about the role of Akt2 in modulating cardiac metabolism, survival, and growth. Here, we utilize murine models with targeted disruption of the akt2 or the akt1 genes to demonstrate that Akt2, but not Akt1, is required for insulin-stimulated 2-[3H]deoxyglucose uptake and metabolism. In contrast, akt2-/- mice displayed normal cardiac growth responses to provocative stimulation, including ligand stimulation of cultured cardiomyocytes, pressure overload by transverse aortic constriction, and myocardial infarction. However, akt2-/- mice were found to be sensitized to cardiomyocyte apoptosis in response to ischemic injury, and apoptosis was significantly increased in the peri-infarct zone of akt2-/- hearts 7 days after occlusion of the left coronary artery. These results implicate Akt2 in the regulation of cardiomyocyte metabolism and survival.

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