Structural and genomic decoding of human and plant myristoylomes reveals a definitive recognition pattern.

An organism's entire protein modification repertoire has yet to be comprehensively mapped. N-myristoylation (MYR) is a crucial eukaryotic N-terminal protein modification. Here we mapped complete Homo sapiens and Arabidopsis thaliana myristoylomes. The crystal structures of human modifier NMT1 complexed with reactive and nonreactive target-mimicking peptide ligands revealed unexpected binding clefts and a modifier recognition pattern. This information allowed integrated mapping of myristoylomes using peptide macroarrays, dedicated prediction algorithms, and in vivo mass spectrometry. Global MYR profiling at the genomic scale identified over a thousand novel, heterogeneous targets in both organisms. Surprisingly, MYR involved a non-negligible set of overlapping targets with N-acetylation, and the sequence signature marks for a third proximal acylation-S-palmitoylation-were genomically imprinted, allowing recognition of sequences exhibiting both acylations. Together, the data extend the N-end rule concept for Gly-starting proteins to subcellular compartmentalization and reveal the main neighbors influencing protein modification profiles and consequent cell fate.

How the Warburg effect supports aggressiveness and drug resistance of cancer cells?

Cancer cells employ both conventional oxidative metabolism and glycolytic anaerobic metabolism. However, their proliferation is marked by a shift towards increasing glycolytic metabolism even in the presence of O2 (Warburg effect). HIF1, a major hypoxia induced transcription factor, promotes a dissociation between glycolysis and the tricarboxylic acid cycle, a process limiting the efficient production of ATP and citrate which otherwise would arrest glycolysis. The Warburg effect also favors an intracellular alkaline pH which is a driving force in many aspects of cancer cell proliferation (enhancement of glycolysis and cell cycle progression) and of cancer aggressiveness (resistance to various processes including hypoxia, apoptosis, cytotoxic drugs and immune response). This metabolism leads to epigenetic and genetic alterations with the occurrence of multiple new cell phenotypes which enhance cancer cell growth and aggressiveness. In depth understanding of these metabolic changes in cancer cells may lead to the development of novel therapeutic strategies, which when combined with existing cancer treatments, might improve their effectiveness and/or overcome chemoresistance.

Mechanical stress increases brain amyloid ß, tau, and α-synuclein concentrations in wild-type mice.

INTRODUCTION: Exposure to traumatic brain injury is a core risk factor that predisposes an individual to sporadic neurodegenerative diseases. We provide evidence that mechanical stress increases brain levels of hallmark proteins associated with neurodegeneration. METHODS: Wild-type mice were exposed to multiple regimens of repetitive mild traumatic brain injury, generating a range of combinations of impact energies, frequencies, and durations of exposure. Brain concentrations of amyloid ß 1-42 (Aß1-42), total tau, and α-synuclein were measured by sandwich enzyme-linked immunosorbent assay. RESULTS: There was a highly significant main effect of impact energy, frequency, and duration of exposure on Aß1-42, tau, and α-synuclein levels (P < .001), and a significant interaction between impact energy and duration of exposure for Aß1-42 and tau (P < .001), but not for α-synuclein. DISCUSSION: Dose-dependent and cumulative influence of repetitive mild traumatic brain injury-induced mechanical stress may trigger and/or accelerate neurodegeneration by pushing protein concentration over the disease threshold.

Necessary and sufficient conditions for protocell growth.

We consider a generic protocell model consisting of any conservative chemical reaction network embedded within a membrane. The membrane results from the self-assembly of a membrane precursor and is semi-permeable to some nutrients. Nutrients are metabolized into all other species including the membrane precursor, and the membrane grows in area and the protocell in volume. Faithful replication through cell growth and division requires a doubling of both cell volume and surface area every division time (thus leading to a periodic surface area-to-volume ratio) and also requires periodic concentrations of the cell constituents. Building upon these basic considerations, we prove necessary and sufficient conditions pertaining to the chemical reaction network for such a regime to be met. A simple necessary condition is that every moiety must be fed. A stronger necessary condition implies that every siphon must be either fed, or connected to species outside the siphon through a pass reaction capable of transferring net positive mass into the siphon. And in the case of nutrient uptake through passive diffusion and of constant surface area-to-volume ratio, a sufficient condition for the existence of a fixed point is that every siphon be fed. These necessary and sufficient conditions hold for any chemical reaction kinetics, membrane parameters or nutrient flux diffusion constants.

Mechanical stress models of Alzheimer's disease pathology.

INTRODUCTION: Extracellular accumulation of amyloid-ß protein and intracellular accumulation of tau in brain tissues have been described in animal models of Alzheimer's disease (AD) and mechanical stress-based diseases of different mechanisms, such as traumatic brain injury (TBI), arterial hypertension (HTN), and normal pressure hydrocephalus (NPH). METHODS: We provide a brief overview of experimental models of TBI, HTN, and NPH showing features of tau-amyloid pathology, neuroinflammation, and neuronal loss. RESULTS: "Alzheimer-like" hallmarks found in these mechanical stress-based models were compared with AD features found in transgenic models. DISCUSSION: The goal of this review is, therefore, to build on current concepts of onset and progression of AD lesions. We point to the importance of accumulated mechanical stress in brain as an environmental and endogenous factor that pushes protein deposition and neuronal injury over the disease threshold. We further encourage the development of preventing strategies and drug screening based on mechanical stress models.

Mechanical stress related to brain atrophy in Alzheimer's disease.

INTRODUCTION: The effects related to endogenous mechanical energy in Alzheimer's disease (AD) pathology have been widely overlooked. With the support of available data from literature and mathematical arguments, we hypothesize that brain atrophy in AD could be co-driven by the cumulative impact of the pressure within brain tissues. METHODS: Brain volumetric and physical data in AD and normal aging (NA) were extracted from the literature. Average brain shrinkage and axial deformations were evaluated mathematically. Mechanical stress equivalents related to brain shrinkage were calculated using a conservation law derived from fluid and solid mechanics. RESULTS: Pressure equivalents of 5.92 and 3.43 mm Hg were estimated in AD and in NA, respectively. DISCUSSION: The calculated increments of brain mechanical stress in AD, which could be impacted by marked dampening of arterial pulse waves, may point to the need to expand the focus on the mechanical processes underpinning pathologic aging of the brain.

The metabolic cooperation between cells in solid cancer tumors.

Cancer cells cooperate with stromal cells and use their environment to promote tumor growth. Energy production depends on nutrient availability and O2 concentration. Well-oxygenated cells are highly proliferative and reorient the glucose metabolism towards biosynthesis, whereas glutamine oxidation replenishes the TCA cycle coupled with OXPHOS-ATP production. Glucose, glutamine and alanine transformations sustain nucleotide and fatty acid synthesis. In contrast, hypoxic cells slow down their proliferation, enhance glycolysis to produce ATP and reject lactate which is recycled as fuel by normoxic cells. Thus, glucose is spared for biosynthesis and/or for hypoxic cell function. Environmental cells, such as fibroblasts and adipocytes, serve as food donors for cancer cells, which reject waste products (CO2 , H⁺, ammoniac, polyamines…) promoting EMT, invasion, angiogenesis and proliferation. This metabolic-coupling can be considered as a form of commensalism whereby non-malignant cells support the growth of cancer cells. Understanding these cellular cooperations within tumors may be a source of inspiration to develop new anti-cancer agents.

Filamentation as primitive growth mode?

Osmotic pressure influences cellular shape. In a growing cell, chemical reactions and dilution induce changes in osmolarity, which in turn influence the cellular shape. Using a protocell model relying upon random conservative chemical reaction networks with arbitrary stoichiometry, we find that when the membrane is so flexible that its shape adjusts itself quasi-instantaneously to balance the osmotic pressure, the protocell either grows filamentous or fails to grow. This behavior is consistent with a mathematical proof. This suggests that filamentation may be a primitive growth mode resulting from the simple physical property of balanced osmotic pressure. We also find that growth is favored if some chemical species are only present inside the protocell, but not in the outside growth medium. Such an insulation requires specific chemical schemes. Modern evolved cells such as E. coli meet these requirements through active transport mechanisms such as the phosphotransferase system.

Cell cycle progression is regulated by intertwined redox oscillators.

The different phases of the eukaryotic cell cycle are exceptionally well-preserved phenomena. DNA decompaction, RNA and protein synthesis (in late G1 phase) followed by DNA replication (in S phase) and lipid synthesis (in G2 phase) occur after resting cells (in G0) are committed to proliferate. The G1 phase of the cell cycle is characterized by an increase in the glycolytic metabolism, sustained by high NAD+/NADH ratio. A transient cytosolic acidification occurs, probably due to lactic acid synthesis or ATP hydrolysis, followed by cytosolic alkalinization. A hyperpolarized transmembrane potential is also observed, as result of sodium/potassium pump (NaK-ATPase) activity. During progression of the cell cycle, the Pentose Phosphate Pathway (PPP) is activated by increased NADP+/NADPH ratio, converting glucose 6-phosphate to nucleotide precursors. Then, nucleic acid synthesis and DNA replication occur in S phase. Along with S phase, unpublished results show a cytosolic acidification, probably the result of glutaminolysis occurring during this phase. In G2 phase there is a decrease in NADPH concentration (used for membrane lipid synthesis) and a cytoplasmic alkalinization occurs. Mitochondria hyperfusion matches the cytosolic acidification at late G1/S transition and then triggers ATP synthesis by oxidative phosphorylation. We hypothesize here that the cytosolic pH may coordinate mitochondrial activity and thus the different redox cycles, which in turn control the cell metabolism.

Chemical Schemes for Maintaining Different Compositions Across a Semi-permeable Membrane with Application to Proto-cells.

Osmotic pressure arising from a higher total chemical concentration inside proto-cells is thought to have played a role in the emergence and selection of self-replicating proto-cells. We present two chemical schemes through which different equilibrium compositions can coexist on each side of a semi-permeable membrane. The first scheme relies upon the concept of moieties and associated number of degrees of freedom. The second scheme relies upon the concept of siphons and of pass reaction capable of transferring matter from outside a siphon into it. Using simple example reaction networks, we show that both schemes are compatible with stationary proto-cell growth with up-concentration, but suffer from shortcomings. To alleviate these we propose a third scheme derived from the second one by having the pass reaction catalyzed by the membrane surface instead of occurring in bulk solution. This may have proven an intermediate step before having the pass reaction occurring only when the nutrient crosses the membrane. This suggests an evolutionary path for the emergence of active transport.

Tumor regression with a combination of drugs interfering with the tumor metabolism: efficacy of hydroxycitrate, lipoic acid and capsaicin.

Cellular metabolic alterations are now well described as implicated in cancer and some strategies are currently developed to target these different pathways. In previous papers, we demonstrated that a combination of molecules (namely alpha-lipoic acid and hydroxycitrate, i.e. Metabloc™) targeting the cancer metabolism markedly decreased tumor cell growth in mice. In this work, we demonstrate that the addition of capsaicin further delays tumor growth in mice in a dose dependant manner. This is true for the three animal model tested: lung (LLC) cancer, bladder cancer (MBT-2) and melanoma B16F10. There was no apparent side effect of this ternary combination. The addition of a fourth drug (octreotide) is even more effective resulting in tumor regression in mice bearing LLC cancer. These four compounds are all known to target the cellular metabolism not its DNA. The efficacy, the apparent lack of toxicity, the long clinical track records of these medications in human medicine, all points toward the need for a clinical trial. The dramatic efficacy of treatment suggests that cancer may simply be a disease of dysregulated cellular metabolism.

A graph-theoretic approach for classification and structure prediction of transmembrane ß-barrel proteins.

BACKGROUND: Transmembrane ß-barrel proteins are a special class of transmembrane proteins which play several key roles in human body and diseases. Due to experimental difficulties, the number of transmembrane ß-barrel proteins with known structures is very small. Over the years, a number of learning-based methods have been introduced for recognition and structure prediction of transmembrane ß-barrel proteins. Most of these methods emphasize on homology search rather than any biological or chemical basis. RESULTS: We present a novel graph-theoretic model for classification and structure prediction of transmembrane ß-barrel proteins. This model folds proteins based on energy minimization rather than a homology search, avoiding any assumption on availability of training dataset. The ab initio model presented in this paper is the first method to allow for permutations in the structure of transmembrane proteins and provides more structural information than any known algorithm. The model is also able to recognize ß-barrels by assessing the pseudo free energy. We assess the structure prediction on 41 proteins gathered from existing databases on experimentally validated transmembrane ß-barrel proteins. We show that our approach is quite accurate with over 90% F-score on strands and over 74% F-score on residues. The results are comparable to other algorithms suggesting that our pseudo-energy model is close to the actual physical model. We test our classification approach and show that it is able to reject α-helical bundles with 100% accuracy and ß-barrel lipocalins with 97% accuracy. CONCLUSIONS: We show that it is possible to design models for classification and structure prediction for transmembrane ß-barrel proteins which do not depend essentially on training sets but on combinatorial properties of the structures to be proved. These models are fairly accurate, robust and can be run very efficiently on PC-like computers. Such models are useful for the genome screening.

Normal human melanocytes exposed to chronic insulin and glucose supplementation undergo oncogenic changes and methyl group metabolism cellular redistribution.

Recent epidemiological studies have suggested a link between cancer and pathophysiological conditions associated with hyperinsulinemia. In this report, we address the possible role of insulin exposure in melanocyte transformation. To this aim, normal melanocytes were exposed to chronic insulin and glucose supplementation (twice the standard medium concentration) for at least 3 wk. After 3-wk treatment, melanocytes increased proliferation (doubling time: 2.7 vs. 5.6 days, P < 0.01). After 3-wk treatment or after 3-wk treatment followed by 4-wk reculture in standard medium, melanocytes were able to grow in soft agar colonies. Treated melanocytes had increased DNA content (+8%, P < 0.05), chromosomal aberrations, and modified oncoprotein profile: p-Akt expression increased (+32%, P < 0.01), Akt decreased, and c-Myc increased (+40%, P < 0.05). PP2A protein expression increased (+42, P < 0.05), while PP2A methylation decreased (-42%, P < 0.05), and PP2A activity was reduced (-27%, P < 0.05). PP2A transcription level was increased (ppp2r1a, PP2A subunit A, +44%, P < 0.05). Also, transcriptomic data revealed modifications in insr (insulin receptors, +10%, P < 0.05) and Il8 (inflammation protein, +99%, P < 0.01). Glycolysis was modified with increased transcription of Pgk1 and Hif1a (P < 0.05), decreased transcription of Pfkfb3 (P < 0.05), decreased activity of pyruvate kinase (P < 0.01), and decreased pyruvate cell content as assessed by (1)H-NMR spectroscopy. In addition, methyl group metabolism was altered with decreased global DNA methylation (-51%, P < 0.01), increased cytosolic protein methylation (+18%, P < 0.05), and consistent changes in methylated species on (1)H-NMR spectra. In conclusion, exposure to chronic insulin and glucose supplementation induces oncogenic changes and methyl group metabolism redistribution, which may be a biomarker of transformation.

Expected distance between terminal nucleotides of RNA secondary structures.

In "The ends of a large RNA molecule are necessarily close", Yoffe et al. (Nucleic Acids Res 39(1):292-299, 2011) used the programs RNAfold [resp. RNAsubopt] from Vienna RNA Package to calculate the distance between 5' and 3' ends of the minimum free energy secondary structure [resp. thermal equilibrium structures] of viral and random RNA sequences. Here, the 5'-3' distance is defined to be the length of the shortest path from 5' node to 3' node in the undirected graph, whose edge set consists of edges {i, i + 1} corresponding to covalent backbone bonds and of edges {i, j} corresponding to canonical base pairs. From repeated simulations and using a heuristic theoretical argument, Yoffe et al. conclude that the 5'-3' distance is less than a fixed constant, independent of RNA sequence length. In this paper, we provide a rigorous, mathematical framework to study the expected distance from 5' to 3' ends of an RNA sequence. We present recurrence relations that precisely define the expected distance from 5' to 3' ends of an RNA sequence, both for the Turner nearest neighbor energy model, as well as for a simple homopolymer model first defined by Stein and Waterman. We implement dynamic programming algorithms to compute (rather than approximate by repeated application of Vienna RNA Package) the expected distance between 5' and 3' ends of a given RNA sequence, with respect to the Turner energy model. Using methods of analytical combinatorics, that depend on complex analysis, we prove that the asymptotic expected 5'-3' distance of length n homopolymers is approximately equal to the constant 5.47211, while the asymptotic distance is 6.771096 if hairpins have a minimum of 3 unpaired bases and the probability that any two positions can form a base pair is 1/4. Finally, we analyze the 5'-3' distance for secondary structures from the STRAND database, and conclude that the 5'-3' distance is correlated with RNA sequence length.

A Nested 2-Level Cross-Validation Ensemble Learning Pipeline Suggests a Negative Pressure Against Crosstalk snoRNA-mRNA Interactions in Saccharomyces cerevisiae.

The growing number of RNA-mediated regulation mechanisms identified in the past decades suggests a widespread impact of RNA-RNA interactions. The efficiency of the regulation relies on highly specific and coordinated interactions while simultaneously repressing the formation of opportunistic complexes. However, the analysis of RNA interactomes is highly challenging because of the large number of potential partners, discrepancy of the size of RNA families, and the inherent noise in interaction predictions. We designed a recursive two-step cross-validation pipeline to capture the specificity of noncoding RNA (ncRNA) messenger RNA (mRNA) interactomes. Our method has been designed to detect significant loss or gain of specificity between ncRNA-mRNA interaction profiles. Applied to small nucleolar RNA-mRNA in Saccharomyces cerevisiae, our results suggest the existence of a repression of ncRNA affinities with mRNAs and thus the existence of an evolutionary pressure leveling down such interactions.

Alignment of minisatellite maps based on run-length encoding scheme.

Subsequent duplication events are responsible for the evolution of the minisatellite maps. Alignment of two minisatellite maps should therefore take these duplication events into account, in addition to the well-known edit operations. All algorithms for computing an optimal alignment of two maps, including the one presented here, first deduce the costs of optimal duplication scenarios for all substrings of the given maps. Then, they incorporate the pre-computed costs in the alignment recurrence. However, all previous algorithms addressing this problem are dependent on the number of distinct map units (map alphabet) and do not fully make use of the repetitiveness of the map units. In this paper, we present an algorithm that remedies these shortcomings: our algorithm is alphabet-independent and is based on the run-length encoding scheme. It is the fastest in theory, and in practice as well, as shown by experimental results. Furthermore, our alignment model is more general than that of the previous algorithms, and captures better the duplication mechanism. Using our algorithm, we derive a quantitative evidence that there is a directional bias in the growth of minisatellites of the MSY1 dataset.

PP2A activity is controlled by methylation and regulates oncoprotein expression in melanoma cells: a mechanism which participates in growth inhibition induced by chloroethylnitrosourea treatment.

Protein phosphatase 2A (PP2A), an Akt pathway inhibitor, is considered to be activated by methylation of its catalytic subunit. Also PP2A downregulation was proposed to take part in carcinogenesis. Recently, PP2A activation was shown to be activated in response to DNA damage. To obtain further information on the role of PP2A in tumors and response to DNA damage, we investigated the relationship between PP2A methylation and activity, cell proliferation, Akt activation, c-Myc expression and PTEN activity in B16 melanoma cells untreated and after chloroethylnitrosourea (CENU) treatment. In untreated cells, okadaic acid, an antagonist of PP2A methylation, inhibited PP2A activity, stimulated cell proliferation, increased Akt activation and c-Myc expression. Xylulose-5-phosphate, an agonist of PP2A methylation, increased PP2A activity, decreased cell proliferation, Akt activation and c-Myc expression. However, both PP2A methylation modulators increased PTEN activity. During the response to CENU treatment, PP2A methylation and activity were strongly increased, Akt activation and c-Myc expression were decreased. However PTEN activity was increased. After tumor cell growth recovery, these modifications were moderately decreased. PP2A methylation was quantified and correlated positively with PP2A activity, and negatively with criteria for cell aggressiveness (cell proliferation, Akt activation, c-Myc expression). Based on these data, PP2A methylation status controls PP2A activity and oncoproteins expression and PP2A is strongly activated after CENU treatment thus partly explaining the growth inhibition in response to this agent. It follows that PP2A promethylating agents are potential candidates for anticancer drugs.

transFold: a web server for predicting the structure and residue contacts of transmembrane beta-barrels.

Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts. The cellular location and functional diversity of beta-barrel outer membrane proteins makes them an important protein class. At the present time, very few non-homologous TMB structures have been determined by X-ray diffraction because of the experimental difficulty encountered in crystallizing transmembrane (TM) proteins. The transFold web server uses pairwise inter-strand residue statistical potentials derived from globular (non-outer-membrane) proteins to predict the supersecondary structure of TMB. Unlike all previous approaches, transFold does not use machine learning methods such as hidden Markov models or neural networks; instead, transFold employs multi-tape S-attribute grammars to describe all potential conformations, and then applies dynamic programming to determine the global minimum energy supersecondary structure. The transFold web server not only predicts secondary structure and TMB topology, but is the only method which additionally predicts the side-chain orientation of transmembrane beta-strand residues, inter-strand residue contacts and TM beta-strand inclination with respect to the membrane. The program transFold currently outperforms all other methods for accuracy of beta-barrel structure prediction. Available at http://bioinformatics.bc.edu/clotelab/transFold.

Predicting transmembrane beta-barrels and interstrand residue interactions from sequence.

Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The cellular location and functional diversity of beta-barrel outer membrane proteins (omps) makes them an important protein class. At the present time, very few nonhomologous TMB structures have been determined by X-ray diffraction because of the experimental difficulty encountered in crystallizing transmembrane proteins. A novel method using pairwise interstrand residue statistical potentials derived from globular (nonouter membrane) proteins is introduced to predict the supersecondary structure of transmembrane beta-barrel proteins. The algorithm transFold employs a generalized hidden Markov model (i.e., multitape S-attribute grammar) to describe potential beta-barrel supersecondary structures and then computes by dynamic programming the minimum free energy beta-barrel structure. Hence, the approach can be viewed as a "wrapping" component that may capture folding processes with an initiation stage followed by progressive interaction of the sequence with the already-formed motifs. This approach differs significantly from others, which use traditional machine learning to solve this problem, because it does not require a training phase on known TMB structures and is the first to explicitly capture and predict long-range interactions. TransFold outperforms previous programs for predicting TMBs on smaller (

The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect.

To better understand the energetic status of proliferating cells, we have measured the intracellular pH (pHi) and concentrations of key metabolites, such as adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP) in normal and cancer cells, extracted from fresh human colon tissues. Cells were sorted by elutriation and segregated in different phases of the cell cycle (G0/G1/S/G2/M) in order to study their redox (NAD, NADP) and bioenergetic (ATP, pHi) status. Our results show that the average ATP concentration over the cell cycle is higher and the pHi is globally more acidic in normal proliferating cells. The NAD+/NADH and NADP+/NADPH redox ratios are, respectively, five times and ten times higher in cancer cells compared to the normal cell population. These energetic differences in normal and cancer cells may explain the well-described mechanisms behind the Warburg effect. Oscillations in ATP concentration, pHi, NAD+/NADH, and NADP+/NADPH ratios over one cell cycle are reported and the hypothesis addressed. We also investigated the mitochondrial membrane potential (MMP) of human and mice normal and cancer cell lines. A drastic decrease of the MMP is reported in cancer cell lines compared to their normal counterparts. Altogether, these results strongly support the high throughput aerobic glycolysis, or Warburg effect, observed in cancer cells.