Peroxiredoxins (Prxs) are a widespread and highly expressed family of cysteine-based peroxidases that react very rapidly with H 2 O 2 , organic peroxides, and peroxynitrite. Correct subfamily classification has been problematic since Prx subfamilies are frequently not correlated with phylogenetic distribution and diverge in their preferred reductant, oligomerization state, and tendency towards overoxidation. We have developed a method that uses the Deacon Active Site Profiler (DASP) tool to extract functional site profiles from structurally characterized proteins, to computationally define subfamilies, and to identify new Prx subfamily members from GenBank(nr). For the 58 literature-defined Prx test proteins, 57 were correctly assigned and none were assigned to the incorrect subfamily. The >3500 putative Prx sequences identified were then used to analyze residue conservation in the active site of each Prx subfamily. Our results indicate that the existence and location of the resolving cysteine varies in some subfamilies (e.g. Prx5) to a greater degree than previously appreciated and that interactions at the A interface (common to Prx5, Tpx and higher order AhpC/Prx1 structures) are important for stabilization of the correct active site geometry. Interestingly, this method also allows us to further divide the AhpC/Prx1 into four groups that are correlated with functional characteristics. The DASP method provides more accurate subfamily classification than PSI-BLAST for members of the Prx family and can now readily be applied to other large protein families.* Corresponding author: Wake Forest University, Office of the Dean of the College, Winston-Salem, NC 27109, phone: 336-758-5311, fetrowjs@wfu.edu. AUTHOR SUMMARY Genome sequencing projects have resulted in tremendous quantities of sequence information, but experimental characterization of protein function has been performed on only a small fraction of sequences. Although numerous computational methods exist that provide functional classification for many uncharacterized proteins, misannotation is a significant problem, since most sequencefocused methods are unable to distinguish the features of individual subfamilies. Our recently developed method called Deacon Active Site Profiling (DASP) is able to extract the features located near the functional site of structurally characterized proteins and utilize this information to identify other proteins in the sequence database that share similar functional site characteristics. In this paper, we used this method to analyze the widely distributed and moderately well-characterized peroxiredoxin protein family; family members detoxify hydrogen peroxide and other oxidized molecules in the cell. We identified over 3500 putative peroxiredoxin sequences from the sequence database and classified them into one of six subfamilies. Subfamily searches using DASP were highly specific and allowed identification of key features at the active site of each subfamily, providing a number of experimentally testable hypotheses. T...
Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines.
PREX (http://www.csb.wfu.edu/prex/) is a database of currently 3516 peroxiredoxin (Prx or PRDX) protein sequences unambiguously classified into one of six distinct subfamilies. Peroxiredoxins are a diverse and ubiquitous family of highly expressed, cysteine-dependent peroxidases that are important for antioxidant defense and for the regulation of cell signaling pathways in eukaryotes. Subfamily members were identified using the Deacon Active Site Profiler (DASP) bioinformatics tool to focus in on functionally relevant sequence fragments surrounding key residues required for protein activity. Searches of this database can be conducted by protein annotation, accession number, PDB ID, organism name or protein sequence. Output includes the subfamily to which each classified Prx belongs, accession and GI numbers, genus and species and the functional site signature used for classification. The query sequence is also presented aligned with a select group of Prxs for manual evaluation and interpretation by the user. A synopsis of the characteristics of members of each subfamily is also provided along with pertinent references.
Sulfenic acids, formed as transient intermediates during the reaction of cysteine residues with peroxides, play significant roles in enzyme catalysis and regulation, and are also involved in the redox regulation of transcription factors and other signaling proteins. Therefore, interest in the identification of protein sulfenic acids has grown substantially in the past few years. Dimedone, which specifically traps sulfenic acids, has provided the basis for the synthesis of a novel group of compounds that derivatize 1,3-cyclohexadione, a dimedone analogue, with reporter tags such as biotin for affinity capture and fluorescent labels for visual detection. These reagents allow identification of the cysteine sites and proteins that are sensitive to oxidation and permit identification of the cellular conditions under which such oxidations occur. We have shown that these compounds are reactive and specific toward sulfenic acids and that the labeled proteins can be detected at high sensitivity using gel analysis or mass spectrometry. Here, we further characterize these reagents, showing that the DCP-Bio1 incorporation rates into three sulfenic acid containing proteins, papaya papain, Escherichia coli fRMsr, and the Salmonella typhimurium peroxiredoxin AhpC, are significantly different and, in the case of fRMsr, are unaffected by changes in buffer pH from 5.5 and 8.0. We also provide protocols to label protein sulfenic acids in cellular proteins, either by in situ labeling of intact cells or by labeling at the time of lysis. We show that the addition of alkylating reagents and catalase to the lysis buffer is critical in preventing the formation of sulfenic acid subsequent to cell lysis. Data presented herein also indicate that the need to standardize, as much as possible, the protein and reagent concentrations during labeling. Finally, we introduce several new test or control proteins that canbeusedtoevaluate labeling procedures and efficiencies.
Lysophosphatidic acid (LPA) is a growth factor for many cells including prostate and ovarian cancer-derived cell lines. LPA stimulates H2O2 production which is required for growth. However, there are significant gaps in our understanding of the spatial and temporal regulation of H2O2-dependent signaling and the way in which signals are transmitted following receptor activation. Herein, we describe the use of two reagents, DCP-Bio1 and DCP-Rho1, to evaluate the localization of active protein oxidation after LPA stimulation by detection of nascent protein sulfenic acids. We found that LPA stimulation causes internalization of LPA receptors into early endosomes that contain NADPH oxidase components and are sites of H2O2 generation. DCP-Rho1 allowed visualization of sulfenic acid formation, indicative of active protein oxidation, which was stimulated by LPA and decreased by an LPA receptor antagonist. Protein oxidation sites colocalized with LPAR1 and the endosomal marker EEA1. Concurrent with the generation of these redox signaling-active endosomes (redoxosomes) is the H2O2- and NADPH oxidase-dependent oxidation of Akt2 and PTP1B detected using DCP-Bio1. These new approaches therefore enable detection of active, H2O2-dependent protein oxidation linked to cell signaling processes. DCP-Rho1 may be a particularly useful protein oxidation imaging agent enabling spatial resolution due to the transient nature of the sulfenic acid intermediate it detects.
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