Robust determination of differential abundance in shotgun proteomics using nonparametric statistics.

Label-free shotgun mass spectrometry enables the detection of significant changes in protein abundance between different conditions. Due to often limited cohort sizes or replication, large ratios of potential protein markers to number of samples, as well as multiple null measurements pose important technical challenges to conventional parametric models. From a statistical perspective, a scenario similar to that of unlabeled proteomics is encountered in genomics when looking for differentially expressed genes. Still, the difficulty of detecting a large fraction of the true positives without a high false discovery rate is arguably greater in proteomics due to even smaller sample sizes and peptide-to-peptide variability in detectability. These constraints argue for nonparametric (or distribution-free) tests on normalized peptide values, thus minimizing the number of free parameters, as well as for measuring significance with permutation testing. We propose such a procedure with a class-based statistic, no parametric assumptions, and no parameters to select other than a nominal false discovery rate. Our method was tested on a new dataset which is available via ProteomeXchange with identifier PXD006447. The dataset was prepared using a standard proteolytic digest of a human protein mixture at 1.5-fold to 3-fold protein concentration changes and diluted into a constant background of yeast proteins. We demonstrate its superiority relative to other approaches in terms of the realized sensitivity and realized false discovery rates determined by ground truth, and recommend it for detecting differentially abundant proteins from MS data.

Two-domain analysis of JmjN-JmjC and PHD-JmjC lysine demethylases: Detecting an inter-domain evolutionary stress.

Residues at different positions of a multiple sequence alignment sometimes evolve together, due to a correlated structural or functional stress at these positions. Co-evolution has thus been evidenced computationally in multiple proteins or protein domains. Here, we wish to study whether an evolutionary stress is exerted on a sequence alignment across protein domains, i.e., on longer sequence separations than within a single protein domain. JmjC-containing lysine demethylases were chosen for analysis, as a follow-up to previous studies; these proteins are important multidomain epigenetic regulators. In these proteins, the JmjC domain is responsible for the demethylase activity, and surrounding domains interact with histones, DNA or partner proteins. This family of enzymes was analyzed at the sequence level, in order to determine whether the sequence of JmjC-domains was affected by the presence of a neighboring JmjN domain or PHD finger in the protein. Multiple positions within JmjC sequences were shown to have their residue distributions significantly altered by the presence of the second domain. Structural considerations confirmed the relevance of the analysis for JmjN-JmjC proteins, while among PHD-JmjC proteins, the length of the linker region could be correlated to the residues observed at the most affected positions. The correlation of domain architecture with residue types at certain positions, as well as that of overall architecture with protein function, is discussed. The present results thus evidence the existence of an across-domain evolutionary stress in JmjC-containing demethylases, and provide further insights into the overall domain architecture of JmjC domain-containing proteins.

Identification of family determining residues in Jumonji-C lysine demethylases: A sequence-based, family wide classification.

Histone post-translational modifications play a critical role in the regulation of gene expression. Methylation of lysines at N-terminal tails of histones has been shown to be involved in such regulation. While this modification was long considered to be irreversible, two different classes of enzymes capable of carrying out the demethylation of histone lysines were recently identified: the oxidases, such as LSD1, and the oxygenases (JmjC-containing). Here, a family-wide analysis of the second of these classes is proposed, with over 300 proteins studied at the sequence level. We show that a correlated evolution analysis yields some position/residue pairs which are critical at comparing JmjC sequences and enables the classification of JmjC domains into five families. A few positions appear more frequently among conditions, such as positions 23 (directly C-terminal to the second iron ligand), 24, 252 and 253 (directly N-terminal to a conserved Asn). Implications of family conditions are studied in detail on PHF2, revealing the meaningfulness of the sequence-derived conditions at the structural level. These results should help obtain insights on the diversity of JmjC-containing proteins solely by considering some of the amino acids present in their JmjC domain.

A Structure-Based Classification of Class A ß-Lactamases, a Broadly Diverse Family of Enzymes.

For medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A ß-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative ß-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A ß-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum ß-lactamases, extended-spectrum ß-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of ß-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking.

Identification of family-determining residues in PHD fingers.

Histone modifications are fundamental to chromatin structure and transcriptional regulation, and are recognized by a limited number of protein folds. Among these folds are PHD fingers, which are present in most chromatin modification complexes. To date, about 15 PHD finger domains have been structurally characterized, whereas hundreds of different sequences have been identified. Consequently, an important open problem is to predict structural features of a PHD finger knowing only its sequence. Here, we classify PHD fingers into different groups based on the analysis of residue-residue co-evolution in their sequences. We measure the degree to which fixing the amino acid type at one position modifies the frequencies of amino acids at other positions. We then detect those position/amino acid combinations, or 'conditions', which have the strongest impact on other sequence positions. Clustering these strong conditions yields four families, providing informative labels for PHD finger sequences. Existing experimental results, as well as docking calculations performed here, reveal that these families indeed show discrepancies at the functional level. Our method should facilitate the functional characterization of new PHD fingers, as well as other protein families, solely based on sequence information.

N-Hydroxyguanidines oxidation by a N3S copper-complex mimicking the reactivity of Dopamine beta-Hydroxylase.

N-Aryl-N'-hydroxyguanidines are compounds that display interesting pharmacological properties but their chemical reactivity remains poorly investigated. Some of these compounds are substrates for the heme-containing enzymes nitric-oxide synthases (NOS) and act as reducing co-substrates for the copper-containing enzyme Dopamine beta-Hydroxylase (DBH) [P. Slama, J.L. Boucher, M. Réglier, Biochem. Biophys. Res. Commun. 316 (2004) 1081-1087]. DBH catalyses the hydroxylation of the important neurotransmitter dopamine into norepinephrine in the presence of both molecular oxygen and a reducing co-substrate. Although many molecules have been used as co-substrates for DBH, their interaction at the active site of DBH and their role in mechanism are not clearly characterized. In the present paper, we have used a water-soluble copper-N(3)S complex that mimics the Cu(B) site of DBH, and aromatic N-hydroxyguanidines as reducers to address this question. N-Aryl-N'-hydroxyguanidines readily reduced copper(II) to Cu(I) and were oxidized into a nitrosoamidine as previously observed in reactions performed with purified DBH. These data describe for the first time the reactivity of N-aryl-N'-hydroxyguanidines with a water-soluble copper(II) complex and help to understand the interaction of co-substrates with copper at the active site of DBH.

Detection of protein catalytic residues at high precision using local network properties.

BACKGROUND: Identifying the active site of an enzyme is a crucial step in functional studies. While protein sequences and structures can be experimentally characterized, determining which residues build up an active site is not a straightforward process. In the present study a new method for the detection of protein active sites is introduced. This method uses local network descriptors derived from protein three-dimensional structures to determine whether a residue is part of an active site. It thus does not involve any sequence alignment or structure similarity to other proteins. A scoring function is elaborated over a set of more than 220 proteins having different structures and functions, in order to detect protein catalytic sites with a high precision, i.e. with a minimal rate of false positives. RESULTS: The scoring function was based on the counts of first-neighbours on side-chain contacts, third-neighbours and residue type. Precision of the detection using this function was 28.1%, which represents a more than three-fold increase compared to combining closeness centrality with residue surface accessibility, a function which was proposed in recent years. The performance of the scoring function was also analysed into detail over a smaller set of eight proteins. For the detection of 'functional' residues, which were involved either directly in catalytic activity or in the binding of substrates, precision reached a value of 72.7% on this second set. These results suggested that our scoring function was effective at detecting not only catalytic residues, but also any residue that is part of the functional site of a protein. CONCLUSION: As having been validated on the majority of known structural families, this method should prove useful for the detection of active sites in any protein with unknown function, and for direct application to the design of site-directed mutagenesis experiments.

Aromatic N-hydroxyguanidines as new reduction cosubstrates for dopamine beta-hydroxylase.

Conversion of neurotransmitter dopamine into norepinephrine is catalyzed by dopamine beta-hydroxylase (DbH). The reaction requires the presence of both molecular oxygen and a reducing cosubstrate, the assumed physiological cosubstrate being ascorbic acid. We have investigated the ability of a new family of molecules, N-aryl-N'-hydroxyguanidines, to serve as cosubstrates for DbH. N-(4-Methoxyphenyl)-N'-hydroxyguanidine proved to be an efficient reducing agent for DbH. The complete N-hydroxyguanidine moiety was required for activity, as any modification of this function resulted in non-cosubstrate compounds. Moreover, analysis of the products formed from N-(4-methoxyphenyl)-N'-hydroxyguanidine showed that the main oxidation product was a nitrosoimine. Modification of the aromatic para-substituent evidenced an influence of its electronic properties on the catalytic activity whereas steric factors seemed less important. In addition, changing the methoxy-substituent from the para- to the ortho-position led to an inactive compound. Our results demonstrate that N-aryl-N'-hydroxyguanidines are new efficient reducing cosubstrates for DbH and prove that specific interactions with the reducing cosubstrate do take place at the active site of the enzyme.

Copper-containing monooxygenases: enzymatic and biomimetic studies of the O-atom transfer catalysis.

This review reports our recent studies or the mechanism of O-atom transfer to a benzylic C-H bond promoted by Dopamine beta-Hydroxylase (DBH) and its biomimetic models. We demonstrate that it is possible to carry out parallel and comparative studies on this enzyme (DBH) and its biomimetic models with the same substrate: 2-aminoindane (3). It was chosen because its two stereogenic centers, both in benzylic positions, make it very powerful for studying the stereochemistry of an O-atom transfer reaction. DBH-catalyzed hydroxylation of 3 produced exclusively 14% of trans-(1S,2S)-2-amino-1-indanol (4) (93% ee). Studies with stereospecifically deuterium-labeled 2-aminoindanes (1R,2S)-3b and (1S,2S)-3a showed that the formation of 4 was the rcsult of an overall process with retention of configuration where an O-atom is stereospecifically inserted in the trans pro-S position of the substrate. With copper(I) and (II) complexes of IndPY2 ligands we studied the reaction with dioxygen and observed an O-atom transfer to a benzylic C-H bond which was performed in the same manner as that of DBH. With the deuterium-labeled cis-2-d-IndPY2 ligand, we demonstrated that the reaction occurs by a stereospecific process with retention of configuration. In both cases (enzymatic vs. biomimetic) the O-atom transfers occur in a two-step process involving radical intermediates.