Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys⁷³ only after the formation of a Cys³²-Cys³⁵ disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1^C32S/C35S (a mutant Trx1 with both Cys³² and Cys³⁵ replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys¹⁷³ and Cys⁸³ and protects it from H₂O₂-induced overoxidation. Functionally, we found that Cys⁷³-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation.Nitric oxide (NO) is an important second messenger for signal transduction in cells. The production of cGMP by guanylyl cyclase, enabled by the binding of NO onto heme, is considered the primary mechanism responsible for the plethora of functions exerted by NO (1). However, S-nitrosylation, the covalent addition of the NO moiety onto cysteine thiols, is increasingly recognized as an important post-translational modification for regulating protein functions (for reviews, see Refs. 2 and 3). S-Nitrosylation is dynamic, reversible, site-specific, and modulated by selected cellular stimuli (4–7). With improved detection sensitivity, an increasing number of S-nitrosylated proteins have been identified by proteomics technologies (5, 8–13). Among the known modified proteins, nitrosylation occurs only on selected cysteines (4, 6, 14–17). Non-enzymatic mechanisms proposed to determine S-nitrosylation specificity include the availability of specific NO donors and protein microenvironments that stabilize the pK_a of acidic target cysteines (18). Furthermore, several enzymes, including hemoglobin (19, 20), superoxide dismutase 1 (21, 22), S-nitrosoglutathione reductase (23–25), and protein-disulfide isomerase (26), have been shown to possess either transnitrosylase or denitrosylase activities. However, an enzymatic system that governs site-specific transnitrosylation and denitrosylation, analogous to the kinase/phosphatase paradigm for regulating protein phosphorylation, has remained largely uncharacterized.Trx1¹ is an important antioxidant protein with protein reductase activity (27, 28). It has been characterized as an antiapoptotic protein because of its ability to suppress proapoptotic proteins, including apoptosis signal-regulating kinase 1 via disulfide reduction and Casp3 via transnitrosylation of Cys¹⁶³ (14, 29). Conversely, Trx1 can denitrosylate Casp3 at Cys¹⁶³, resulting in Casp3 activation (7). Trx1 appears to govern site-specific reversible nitrosylation of selected protein targets (14, 15), but what are the underlying mechanisms that regulate Trx1 transnitrosylation and denitrosylation activities? Are there additional Trx1-mediated transnitrosylation or denitrosylation targets that have not yet been identified? In this study, we used ESI-Q-TOF mass spectrometry (MS) to analyze the nitrosylation of Trx1 and a Casp3 peptide (Casp3p) under different redox conditions. Because of the labile nature of the S–NO bond, direct identification of S-nitrosylated proteins and their specific nitrosylation sites by MS remains challenging (8). A biotin switch method that is based on the derivatization of protein S–NO with a biotinylating agent is typically used for such analyses (8). However, like any indirect method, both false positive and negative identifications have been reported (30). Recently, we developed a method for direct analysis of protein S-nitrosylation by ESI-Q-TOF MS without prior chemical derivatization (31). Here we applied the same technique to determine the regulation of Trx1 by stepwise oxidative and nitrosative modifications of distinct cysteines and its subsequent ability to transnitrosylate target proteins. Nitrosative modification at Cys⁷³ of Trx1 cannot occur without prior attenuation of the Trx1 disulfide reductase and denitrosylase activities via either disulfide bond formation between Cys³² and Cys³⁵ or their mutation to serines. This is a key observation that has never been previously reported. Consequently, we designed a proteomics approach and discovered over 40 putative Trx1 transnitrosylation target proteins. We further characterized the Trx1 transnitrosylation proteome and identified three consensus motifs surrounding the putative Trx1 transnitrosylation sites, suggesting a protein-protein interaction mechanism for determining transnitrosylation specificity.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

A Substrate Trapping Approach Identifies Proteins Regulated by Reversible S-nitrosylation

Protein S-nitrosylation, the nitric oxide-mediated posttranslational modification of cysteine residues, has emerged as an important regulatory mechanism in diverse cellular processes. Yet, knowledge about the S-nitrosoproteome in different cell types and cellular contexts is still limited and many questions remain regarding the precise roles of protein S-nitrosylation and denitrosylation. Here we present a novel strategy to identify reversibly nitrosylated proteins. Our approach is based on nitrosothiol capture and enrichment using a thioredoxin trap mutant, followed by protein identification by mass spectrometry. Employing this approach, we identified more than 400 putative nitroso-proteins in S-nitrosocysteine-treated human monocytes and about 200 nitrosylation substrates in endotoxin and cytokine-stimulated mouse macrophages. The large majority of these represent novel nitrosylation targets and they include many proteins with key functions in cellular homeostasis and signaling. Biochemical and functional experiments in vitro and in cells validated the proteomic results and further suggested a role for thioredoxin in the denitrosylation and activation of inducible nitric oxide synthase and the protein kinase MEK1. Our findings contribute to a better understanding of the macrophage S-nitrosoproteome and the role of thioredoxin-mediated denitrosylation in nitric oxide signaling. The approach described here may prove generally useful for the identification and exploration of nitroso-proteomes under various physiological and pathophysiological conditions.Protein S-nitrosylation, the covalent addition of a nitric oxide (NO)¹ group to a cysteine thiol, constitutes a widespread regulatory mechanism involved in various biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis (1–4). S-nitrosylation is known to modulate the functional properties of a large number of proteins and thereby influence normal cell function and emerging evidence implicates aberrant protein S-nitrosylation in multiple pathological conditions, including cardiovascular disease, neurodegeneration, and cancer (5, 6). Although significant advances have been made in the field of S-nitrosylation, there is still limited knowledge regarding the constituents of the proteome that become nitrosylated (that is, the nitrosoproteome) across different cell types and conditions. Therefore, much remains unknown about the specific roles and functional significance of S-nitrosylation in cellular function and disease. In addition, there is a need to better understand the mechanisms and consequences of denitrosylation, both for individual proteins and on a systems level.Protein denitrosylation is substantially mediated by two cellular denitrosylating systems, namely the glutathione and S-nitrosoglutathione reductase (GSH/GSNOR) and the thioredoxin and thioredoxin reductase (Trx/TrxR) systems, with the latter representing a direct mechanism of protein denitrosylation (7–11). Trx/TrxR-mediated denitrosylation has been specifically linked to several cellular processes, including apoptosis (7), cell adhesion (12), exocytosis (13) and heme protein maturation (14). Despite recent progress in characterizing protein nitrosylation and denitrosylation, the dynamic cellular nitrosoproteome remains relatively unexplored, particularly under physiologically relevant conditions. This may be in part because of methodological challenges inherent to the proteomic analysis of S-nitrosylation and denitrosylation (See Discussion and (15–17)).The disulfide and nitrosothiol (SNO) reductase activities of Trx depend on a highly conserved Cys-Gly-Pro-Cys active site (8, 18). Recent evidence suggests that, similar to reduction of disulfides, SNO reduction occurs in a two-step mechanism (7, 8). First, the more N-terminal cysteine (Cys32 in human Trx1) attacks the sulfur atom on the SNO moiety of the substrate protein, thereby displacing NO (formally, NO⁻) and generating an intermolecular disulfide between Trx and the substrate. The second step entails an intramolecular attack by the second active site cysteine (Cys35, known as the “resolving cysteine”) on the mixed disulfide intermediate, thus releasing the reduced target protein and the oxidized Trx. The normally transient disulfide intermediate formed in the first step is stabilized in a reaction that involves a Trx mutant that lacks the resolving cysteine. This so-called “trap mutant” has been employed in the identification of disulfide targets of Trx in several cell systems (19–22). However, the utility and value of such a trapping approach in the context of nitrosylation proteomics has not been evaluated.In this study we adapted the Trx trapping strategy for global profiling of cellular nitrosylation and denitrosylation processes. Using this approach, we report the identification of hundreds of potential new nitrosylated targets in monoctyes and macrophages, followed by validation using biochemical and functional assays. The findings presented herein greatly expand our knowledge of the monocyte and macrophage nitrosoproteome and suggest multiple roles for nitrosylation and denitrosylation in macrophage activation and function. The approach employed in this study may be applied to exploring nitrosoproteomes in different cells and under various physiological and pathological conditions.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]