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]     

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]     

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[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]     

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen''s ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host''s stomach. Although Hp''s glycans are linked to pathogenesis, Hp''s glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp''s glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp''s azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.Helicobacter pylori (Hp)¹ infection poses a significant health risk to humans worldwide. The Gram-negative, pathogenic bacterium Hp colonizes the gastric tract of more than 50% of humans (1). Approximately 15% of infected individuals develop duodenal ulcers and 1% of infected individuals develop gastric cancer (2). Current treatment to clear infection requires “triple therapy” (3), a combination of multiple antibiotics that is often associated with negative side effects (4). Because of poor patient compliance and the evolution of antibiotic resistance, existing antibiotics are no longer effective at eradicating Hp infection (4). New treatment methods are needed to eliminate Hp from the human gastric tract.Recent work has focused on gaining insights into the pathogenesis of Hp to aid the development of new treatments. The most recent findings in this area have conclusively revealed that glycosylation of proteins in Hp is required for pathogenesis. Hp use complex flagella, comprised of flagellin proteins, to navigate the host''s gastric mucosa (5, 6). The flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, found exclusively in mucosal-associated pathogens (Hp (7), Campylobacter jejuni (8) and Pseudomonas aeruginosa (9)). This modification is absolutely essential for the formation of functional flagella on Hp (7, 10). Deletion of any one of the enzymes in the pseudaminic acid biosynthetic pathway results in Hp that lack flagella, are nonmotile, and are unable to colonize the host''s stomach (7). Although pseudaminic acid is critical for Hp virulence, it is absent from humans (11, 12). Therefore, insights into Hp''s pathogenesis have revealed that Hp''s glycan pseudaminic acid is a bona fide target of therapeutic intervention. This is one of a number of examples linking protein glycosylation to virulence in medically significant bacterial pathogens (13, 14).Despite these findings, Hp''s glycome remains poorly understood overall. Only the two flagellin glycoproteins have been firmly characterized in Hp (7) to date. Nine other candidate glycoproteins have been identified in Hp, but their glycosylation status has not been biochemically confirmed (15). The relative paucity of information regarding Hp''s glycoproteins is due in part to the previously held belief that protein glycosylation could not occur in bacteria (13, 16, 17). However, even after Szymanski (18, 19), Koomey (20), Guerry (21), Logan (7), Comstock and others (13, 16, 17) disproved this belief by firmly establishing the synthesis of glycoproteins in bacteria, the study of bacterial glycoproteins has presented unique challenges for analytical study (14, 22). For example, the unusual structures of bacterial glycans, which often contain amino- and deoxy-carbohydrates exclusively found in bacteria (12, 23–25), hampers their identification using existing tools. Though methods such as the use of glycan-binding reagents (20, 24, 26, 27) and periodic acid/hydrazide glycan labeling (15) have successfully detected glycoproteins in a range of bacteria, they present limitations. Glycan binding-based methods are often limited because of the unavailability of lectins or antibodies with binding specificity for glycosylated proteins in the bacteria of interest (14, 22). Periodic acid/hydrazide-based labeling is plagued by a lack of specificity for glycosylated proteins (15). Thus, an efficient and robust approach to discover Hp''s glycoproteins is needed.In previous work, we established that the chemical technique known as metabolic oligosaccharide engineering (MOE), which was developed by Bertozzi (28, 29), Reutter (30), and others for the study of mammalian glycoproteins, is a powerful approach to label and detect Hp''s glycoproteins (31). Briefly, Hp metabolically processes the unnatural, azide-containing sugar peracetylated N-azidoacetylglucosamine (Ac₄GlcNAz) (32), an analog of the common metabolic precursor N-acetylglucosamine (GlcNAc), into cellular glycoproteins (Fig. 1). Elaboration of azide-labeled glycoproteins via Staudinger ligation (33) with a phosphine probe conjugated to a FLAG peptide (Phos-FLAG) (34) followed by visualization with an anti-FLAG antibody (Fig. 1) revealed a glycoprotein fingerprint containing a large number of as-yet unidentified Hp glycoproteins that merit further investigation (31).Open in a separate windowFig. 1.Metabolic oligosaccharide engineering facilitates labeling and detection of Hp''s glycoproteins. Supplementation of Hp with Ac₄GlcNAz leads to metabolic labeling of Hp''s N-linked and O-linked glycoproteins with azides. Azide-modified glycoproteins covalently labeled with Phos-FLAG can be detected via Western blot analysis with anti-FLAG antibody to yield Hp''s glycoprotein fingerprint, which contains a large number of as-yet unidentified glycoproteins.Here we describe a glycoproteomic identification strategy for the selective detection, isolation, and discovery of Hp''s glycoproteins. In particular, we demonstrate that glycan metabolic labeling coupled with mass spectrometry analysis is an efficient and robust chemical approach to identify novel glycoproteins in Hp. This work characterizes glycosylated virulence factors in Hp, thus opening the door to new vaccination and antibiotic therapies to eradicate Hp infection. Broadly, this work validates metabolic oligosaccharide engineering as a complementary method to discover bacterial glycoproteins.