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...

Objective To better understand the contribution of age to the development of osteoarthritis (OA). Methods Surgical destabilization of the medial meniscus (DMM) was used to model OA in 12 week-old and 12 month-old male C57/BL6 mice. OA severity was evaluated histologically. RNA used for microarrays and real-time PCR was isolated from joint tissue collected from the medial side of the joint, including cartilage, meniscus, subchondral bone, and joint capsule with synovium. Computational analysis was used to identify patterns of gene expression and immunohistochemistry to evaluate tissue distribution of selected proteins. Results OA was more severe in older mice than young. Only 55 genes showed a similar expression with DMM-induced OA in the two age groups while 493 genes showed differential expression, the majority having increased expression in older mice. Functional categories for similarly expressed genes included extracellular matrix and cell adhesion related genes; differentially expressed genes included muscle structure and development and immunoglobulin domain genes. Comparison of expression in the sham control joints revealed an age-related decrease in matrix gene expression and an increase in immune and defense response genes. IL-33 was present in multiple joint tissue cells while CCL21 was more localized to chondrocytes and meniscal cells. Periostin was found in the extracellular matrix of cartilage and meniscus. Conclusion Age affects both the basal pattern of gene expression in joint tissues and the response to surgically-induced OA. Examining tissue from the joint beyond only cartilage revealed novel genes and proteins that would be important to consider in OA.

Cysteine sulfenic acid formation in proteins results from the oxidative modification of susceptible cysteine residues by hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. This species represents a biologically-significant modification occurring during oxidant signaling or oxidative stress and it can modulate protein function. Most methods to identify such oxidatively-modified proteins rely on monitoring the loss of one or more thiol group(s) or on selective labeling of nascent thiol groups following reduction of oxidized proteins. Our previous work reported the direct labeling of these chemically distinct modifications with a dimedone analogue, 1,3-cyclohexadione, to which a linker and functional group (an alcohol) had been added; further addition of a fluorescent isatoic acid or methoxycoumarin reporter allowed detection of the incorporated tag by fluorescence techniques [Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. (2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjug Chem 16, 1624Chem 16, -1628. We have now expanded our arsenal of tagging reagents to include two fluorescein-, two rhodamineand three biotin-conjugated probes based on the original approach. The new tools provide readily detectable fluorescent and affinity probes to identify sulfenic acid modifications in proteins and have been used in subsequent mass spectrometric analyses to confirm covalent attachment of the conjugates and directly determine the site of modification. Keywordscysteine sulfenic acid; reactive oxygen species; oxidized cysteine; papain; peroxiredoxins; peroxide; redox sensor; redox signaling Given the significant role played by formation of cysteine sulfenic acid (S-hydroxycysteine, R-SOH) in the redox regulation of enzymes and transcription regulators (1-3) and its general instability toward protein analytical methods (4), there is a critical need for better reagents to trap and identify these modifications in proteins. Based on a known alkylator of R-SOH, *To whom correspondence should be addressed: Department of Biochemistry, Center for Structural Biology, Medical Center Boulevard, Winston-Salem, NC 27157; Telephone: 336-716-6711 (Poole), 336-758-5774 (King); Fax: 336-777-3242 (Poole), 336-758-4656 (King); E-mail: lbpoole@wfubmc.edu, kingsb@wfu.edu. NIH Public Access Author ManuscriptBioconjug Chem. Author manuscript; available in PMC 2008 November 1. Published in final edited form as:Bioconjug Chem. 2007 ; 18(6): 2004-2017. doi:10.1021/bc700257a. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript dimedone (5,5-dimethyl-1,3-cyclohexanedione), we previously designed, synthesized and validated the use of two fluorescent reagents linked to the reactive core of dimedone, 1,3-cyclohexadione, as detectable markers of R-SOH formation in proteins (5). These reagents were shown to specifically trap only the R-SOH modification in a test protein, AhpC (a cysteine-based peroxidase from bacteria), leaving underivatized the other protein...

Omega (omega-) loops, a nonregular secondary structure found in globular proteins, are characterized by a polypeptide chain that follows a loop-shaped course in three-dimensional space. They do not contain repeating backbone dihedral angles or regular patterns of hydrogen bonding; however, many omega-loops contain a large number of hydrogen bonds, therefore it is not correct to think of omega-loops as structures lacking in hydrogen bonds. omega-Loops are found almost exclusively at the protein surface and exhibit amino acid preferences consistent with this observation. Since the first description of omega-loops in 1986, experiments have been conducted to probe the role of these structures in protein function, stability, and folding. It has become clear that omega-loops are often involved in protein function and molecular recognition. One motif, an omega-loop lid, that is flexible and mobile until substrate or inhibitor is bound and which probably plays a role in one or more steps of enzymatic catalysis, has been described in a variety of enzymes. Because they lack the periodic hydrogen bonding patterns of the regular secondary structures, some omega-loops are well suited for such functional roles in proteins. However, loops with a higher-than-average number of hydrogen bonds or hydrophobic contacts may play roles in protein stability or folding. Rather than determining further geometric definitions of loops, it may be instructional to group them according to their roles in protein structure, i.e., as categories of functional omega-loops, stability omega-loops, and folding omega-loops.

To identify gene products that participate in auxin-dependent lateral root formation, a high temporal resolution, genome-wide transcript abundance analysis was performed with auxin-treated Arabidopsis thaliana roots. Data analysis identified 1246 transcripts that were consistently regulated by indole-3-acetic acid (IAA), partitioning into 60 clusters with distinct response kinetics. We identified rapidly induced clusters containing auxin-response functional annotations and clusters exhibiting delayed induction linked to cell division temporally correlated with lateral root induction. Several clusters were enriched with genes encoding proteins involved in cell wall modification, opening the possibility for understanding mechanistic details of cell structural changes that result in root formation following auxin treatment. Mutants with insertions in 72 genes annotated with a cell wall remodeling function were examined for alterations in IAA-regulated root growth and development. This reverse-genetic screen yielded eight mutants with root phenotypes. Detailed characterization of seedlings with mutations in CELLULASE3/GLYCOSYLHYDROLASE9B3 and LEUCINE RICH EXTENSIN2, genes not normally linked to auxin response, revealed defects in the early and late stages of lateral root development, respectively. The genes identified here using kinetic insight into expression changes lay the foundation for mechanistic understanding of auxin-mediated cell wall remodeling as an essential feature of lateral root development.