Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[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]     

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 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]     

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetyl-CoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen.Post-translational modifications (PTMs)¹ are complex and fundamental mechanisms modulating diverse protein properties and functions, and have been associated with almost all known cellular pathways and disease processes (1, 2). Among the hundreds of different PTMs, acylations at lysine residues, such as acetylation (3–6), malonylation (7, 8), crotonylation (9, 10), propionylation (11–13), butyrylation (11, 13), and succinylation (7, 14–16) are crucial for functional regulations of many prokaryotic and eukaryotic proteins. Because these lysine PTMs depend on the acyl-CoA metabolic intermediates, such as acetyl-CoA (Ac-CoA), succinyl-CoA, and malonyl-CoA, lysine acylation could provide a mechanism to respond to changes in the energy status of the cell and regulate energy metabolism and the key metabolic pathways in diverse organisms (17, 18).Among these lysine PTMs, lysine succinylation is a highly dynamic and regulated PTM defined as transfer of a succinyl group (-CO-CH₂-CH₂-CO-) to a lysine residue of a protein molecule (8). It was recently identified and comprehensively validated in both bacterial and mammalian cells (8, 14, 16). It was also identified in core histones, suggesting that lysine succinylation may regulate the functions of histones and affect chromatin structure and gene expression (7). Accumulating evidence suggests that lysine succinylation is a widespread and important PTM in both eukaryotes and prokaryotes and regulates diverse cellular processes (16). The system-wide studies involving lysine-succinylated peptide immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS/MS) have been employed to analyze the bacteria (E. coli) (14, 16), yeast (S. cerevisiae), human (HeLa) cells, and mouse embryonic fibroblasts and liver cells (16, 19). These succinylome studies have generated large data sets of lysine-succinylated proteins in both eukaryotes and prokaryotes and demonstrated the diverse cellular functions of this PTM. Notably, lysine succinylation is widespread among diverse mitochondrial metabolic enzymes that are involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (19, 20). Thus, lysine succinylation is reported as a functional PTM with the potential to impact mitochondrial metabolism and coordinate different metabolic pathways in human cells and bacteria (14, 19–22).Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a major cause of mortality worldwide and claims more human lives annually than any other bacterial pathogen (23). About one third of the world''s population is infected with Mtb, which leads to nearly 1.3 million deaths and 8.6 million new cases of TB in 2012 worldwide (24). Mtb remains a major threat to global health, especially in the developing countries. Emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Mtb, and also the emergence of co-infection between TB and HIV have further worsened the situation (25–27). Among bacterial pathogens, Mtb has a distinctive life cycle spanning different environments and developmental stages (28). Especially, Mtb can exist in dormant or active states in the host, leading to asymptomatic latent TB infection or active TB disease (29). To achieve these different physiologic states, Mtb developed a mechanism to sense diverse signals from the host and to coordinately regulate multiple cellular processes and pathways (30, 31). Mtb has evolved its metabolic network to both maintain and propagate its survival as a species within humans (32–35). It is well accepted that metabolic network is a central mediator and defining feature of the pathogenicity of Mtb (23, 36–38). Knowledge of the regulation of metabolic pathways used by Mtb during infection is therefore important for understanding its pathogenicity, and can also guide the development of novel drug therapies (39). On the other hand, increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells (14, 19–22). It is tempting to speculate that lysine succinylation may play an important regulatory role in metabolic processes in Mtb. However, to the best of our knowledge, no succinylated protein in Mtb has been identified, presenting a major obstacle to understand the regulatory roles of lysine succinylation in this life-threatening pathogen.In order to fill this gap in our knowledge, we have initiated a systematic study of the identities and functional roles of the succinylated protein in Mtb. Because Mtb H37Rv is the first sequenced Mtb strain (40) and has been extensively used for studies in dissecting the roles of individual genes in pathogenesis (41), it was selected as a test case. We analyzed the succinylome of Mtb H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and render particular enrichment to metabolic process. A large proportion of the succinylation sites are present on proteins in the central metabolism pathway. We further dissected the regulatory role of succinylation on acetyl-CoA synthetase (Acs) via site-specific mutagenesis analysis and molecular dynamics (MD) simulations showed that reversible lysine succinylation could inhibit the activity of Acs. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a deacetylase and as a desuccinylase of Acs in in vitro assays. Together, our findings provide significant insights into the range of functions regulated by lysine succinylation in Mtb.     

Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase

In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.In eukaryotic cells most protein breakdown in the cytosol and nucleus is catalyzed by the 26 S proteasome. This ∼2.5-MDa (1) complex degrades ubiquitin-conjugated and certain non-ubiquitinated proteins in an ATP-dependent manner (2, 3). The 26 S complex is composed of one or two 19 S regulatory particles situated at the ends of the cylindrical 20 S proteasome. Within the 26 S complex, proteins are hydrolyzed in the 20 S proteasome. Tagged substrates, however, first bind to the 19 S regulatory particle, which catalyzes their unfolding and translocation into the 20 S subcomplex (4, 5). The 19 S regulatory particle consists of at least 17 different subunits (1, 6). Nine of these subunits form a “lid,” whereas the other eight subunits, including six ATPases, comprise the base of the 19 S particle. Electron microscopy (7–10) as well as cross-linking experiments (11, 12) have demonstrated that the six homologous ATPases are associated with the α rings of the 20 S particle.Unlike eukaryotes, Archaea and certain eubacteria contain homologous 20 S particles but lack ubiquitin. Their proteasomes degrade proteins in association with a hexameric ATPase ring complex termed PAN (13). PAN appears to be the evolutionary precursor of the 19 S base, predating the coupling of ubiquitination and proteolysis in eukaryotes (14). In addition, PAN recognizes the bacterial targeting sequence ssrA (in analogy to the polyubiquitin conjugates in eukaryotes) and efficiently unfolds and translocates globular substrates, like green fluorescent protein, when tagged with ssrA (15). In both PAN and the 19 S proteasome regulatory complexes, ATP is essential for substrate unfolding and translocation and for opening of the gated channel in the α ring through which substrates enter the 20 S particle (15–17). Because this portal is quite narrow (18–20), only extended polypeptides can enter the 20 S proteasome. Consequently, a globular substrate must be unfolded by the associated ATPase complex to be translocated and digested within the 20 S particle.PAN and the six ATPases found at the base of the 19 S particle are members of the AAA+ superfamily of multimeric ATPases which also includes the ATP-dependent proteases Lon and FtsH and the regulatory components of the bacterial ATP-dependent proteases ClpAP, ClpXP, and HslUV (8, 21). For mechanistic studies of the roles of ATP, the simpler archaeal PAN-20 S system offers many technical advantages over the much more complex 26 S proteasome. For example, prior studies of PAN (17, 22) demonstrated that unfolding of globular substrates (e.g. green fluorescent protein-ssrA) requires ATP hydrolysis. The same was also shown for the Escherichia coli ATP-dependent proteases ClpXP (23) and ClpAP (24). We have also shown that unfolding by PAN can take place on the surface of the ATPase ring in the absence of translocation (15). Thus, unfolding seems to proceed independently from protein translocation into the 20 S proteolytic particle. It is noteworthy that other studies suggest that proteins are unfolded by energy-dependent translocation through the ATPase ring (25, 26). These studies have suggested that the translocation of an unfolded polypeptide from the ATPase into the 20 S core is an active process that is coupled to ATP hydrolysis. A key to underline a detailed molecular mechanism for substrate binding, unfolding, and translocation by the proteasome regulatory ATPase complex is improved understanding of its architecture and the nucleotide-dependent structural transitions that afford these functions.To date we and others have failed to generate micrographs suitable for three-dimensional reconstruction of PAN using single-particle EM analysis. Likewise, structural information regarding the three-dimensional architecture and subunit organization within the 19 S particle is very limited. In fact, high resolution three-dimensional information on the 19 S complex is not yet available. Most knowledge available is based on cross-linking experiments (11, 12) as well as EM structural analysis (7–10), which provided a three-dimensional model outline of the general architecture of the 26 S complex. Unlike the 19 S complex, the structure of the 20 S subcomplex was determined by x-ray crystallography (18, 19). In contrast to the highly homogenous structure of the 20 S complex, the structural heterogeneity and flexibility of the 19 S subcomplex is presumably reflected in multiple conformations, which in turn also contribute to the difficulty in generating a high resolution three-dimensional structural model of the 26 S proteasome. Accordingly, the initial goal of this study was to generate a three-dimensional model of PAN that will allow us to determine its general architecture and to correlate unique conformational transitions within this ATPase with the nucleotide state of the complex (i.e. in the presence of ATPγS, ADP, or in the absence of nucleotides).Smith et al. (27) suggested a general architecture for the PAN-20 S complex based on two-dimensional averaging of a Thermoplasma acidophilum (TA)³ 20 S proteasome and Methanococcus jannaschii (MJ) PAN hybrid complex in the presence of ATPγS. Based on side-view projections of that complex, these authors proposed that PAN assumes an overall structure similar to E. coli HslU (28–30).We realized that although PAN appears heterogeneous in electron micrographs, it does not occupy all possible orientations when adsorbed to carbon-coated electron microscopy (EM) grids, a prerequisite for single particle analysis. This problem was overcome by applying electron tomography in conjunction with a three-dimensional averaging procedure that accounts for the missing wedge in the Fourier space of electron tomograms (31, 32). The three-dimensional model generated revealed an unexpected architecture leading to a possible molecular mechanism describing the function of PAN and presumably the 19 S ATPases.