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Quantitative phosphoproteomics represents a front line for functional proteomics and hence for systems biology. Here we present a new application of the surface-activated chemical ionization (SACI) technology for quantitative phosphoproteomics analysis. The main advantages of SACI-MS technology are high sensitivity, quantitative accuracy and matrix effect reduction, which allow quantitative estimations. METHODS: A SACI-MS approach was used to investigate the quantitative in vivo phosphorylation of the cyclin-dependent kinase inhibitor Sic1, a low-abundance protein of Saccharomyces cerevisiae, which is phosphorylated on Ser201 by casein kinase 2 (CK2) and compared its phosphorylation status in cells growing in two different carbon sources (glucose or ethanol). RESULTS: Our relative quantification indicated that the Sic1-Ser201 phosphorylation level is about 2-fold higher in ethanol- than in glucose-growing cells, proportional to the Sic1 protein level. This finding is coherent with results of western blot analysis using anti-phospho-Ser201-specific antibody, validating the results obtained with this new SACI approach. CONCLUSIONS: The findings presented in this paper indicate that the innovative LC/SACI-MS method, coupled with immunoprecipitation, is a powerful device to obtain quantitative information on the phosphorylation state of low abundance proteins.

Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methalloproteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NG

Neuroglial cells are fundamental for control of brain homeostasis and synaptic plasticity. Decades of pathological and physiological studies have focused on neurons in neurodegenerative disorders, but it is becoming increasingly evident that glial cells play an irreplaceable part in brain homeostasis and synaptic plasticity. Animal models of brain injury and neurodegenerative diseases have largely contributed to current understanding of astrocyte-specific mechanisms participating in brain function and neurodegeneration. Specifically, gliotransmission (presence of glial neurotransmitters, and their receptors and active transporters), trophic support (release, maturation and degradation of neurotrophins) and metabolism (production of lactate and GSH components) are relevant aspects of astrocyte function in neuronal metabolism, synaptic plasticity and neuroprotection. Morpho-functional changes of astrocytes and microglial cells after traumatic or toxic insults to the central nervous system (namely, reactive gliosis) disrupt the complex neuro-glial networks underlying homeostasis and connectivity within brain circuits. Thus, neurodegenerative diseases might be primarily regarded as gliodegenerative processes, in which profound alterations of glial activation have a clear impact on progression and outcomes of neuropathological processes. This review provides an overview of current knowledge of astrocyte functions in the brain and how targeting glial-specific pathways might ultimately impact the development of therapies for clinical management of neurodegenerative disorders.

Great interest is presently given to the analysis of metabolic changes that take place specifically in cancer cells. In this review we summarize the alterations in glycolysis, glutamine utilization, fatty acid synthesis and mitochondrial function that have been reported to occur in cancer cells and in human tumors. We then propose considering cancer as a system-level disease and argue how two hallmarks of cancer, enhanced cell proliferation and evasion from apoptosis, may be evaluated as system-level properties, and how this perspective is going to modify drug discovery. Given the relevance of the analysis of metabolism both for studies on the molecular basis of cancer cell phenotype and for clinical applications, the more relevant technologies for this purpose, from metabolome and metabolic flux analysis in cells by Nuclear Magnetic Resonance and Mass Spectrometry technologies to positron emission tomography on patients, are analyzed. The perspectives offered by specific changes in metabolism for a new drug discovery strategy for cancer are discussed and a survey of the industrial activity already going on in the field is reported.

Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.

The highly dynamic and heterogeneous molecular ensembles characterizing intrinsically disordered proteins (IDP) in solution pose major challenges to the conventional methods for structural analysis. Electrospray ionization-mass spectrometry (ESI-MS) allows direct detection of distinct conformational components, effectively capturing also partially folded and short-lived states. We report the description of two complementary fragments (1-186 and 187-284) of the IDP Sic1, a cyclin-dependent protein kinase inhibitor of yeast Saccharomyces cerevisiae. Structural heterogeneity is noted in both cases, but the two fragments reveal slightly different conformational properties. The results are consistent with previously reported differences between the two protein moieties and corroborate the feasibility of IDP conformational analysis by ESI-MS.

Besides being the favorite carbon and energy source for the budding yeast Sacchromyces cerevisiae, glucose can act as a signaling molecule to regulate multiple aspects of yeast physiology. Yeast cells have evolved several mechanisms for monitoring the level of glucose in their habitat and respond quickly to frequent changes in the sugar availability in the environment: the cAMP/PKA pathways (with its two branches comprising Ras and the Gpr1/Gpa2 module), the Rgt2/Snf3-Rgt1 pathway and the main repression pathway involving the kinase Snf1. The cAMP/PKA pathway plays the prominent role in responding to changes in glucose availability and initiating the signaling processes that promote cell growth and division. Snf1 (the yeast homologous to mammalian AMP-activated protein kinase) is primarily required for the adaptation of yeast cell to glucose limitation and for growth on alternative carbon source, but it is also involved in the cellular response to various environmental stresses. The Rgt2/Snf3-Rgt1 pathway regulates the expression of genes required for glucose uptake. Many interconnections exist between the diverse glucose sensing systems, which enables yeast cells to fine tune cell growth, cell cycle and their coordination in response to nutritional changes.

Bevor sich eine Zelle teilt muss sie ihr gesamtes genetisches Material verdoppeln. Eukaryotische Genome werden von einer Vielzahl von Replikationsstartpunkten, den sogenannten Origins, aus repliziert, die über das gesamte Genome verteilt sind. In dieser Arbeit wird der zugrundeliegende molekulare Mechanismus quantitativ analysiert, der für die nahezu simultane Initiierung der Origins exakt ein Mal pro Zellzyklus verantwortlich ist. Basierend auf umfangreichen experimentellen Studien, wird zunächst ein molekulares regulatorisches Netzwerk rekonstruiert, welches das Binden von Molekülen an die Origins beschreibt, an denen sich schließlich komplette Replikationskomplexe (RKs) bilden. Die molekularen Reaktionen werden dann in ein Differentialgleichungssystem übersetzt. Um dieses mathematische Modell zu parametrisieren, werden gemessene Proteinkonzentrationen als Anfangswerte verwendet, während kinetische Parametersätze in einen Optimierungsverfahren erzeugt werden, in welchem die Dauer, in der sich eine Mindestanzahl von RKs gebildet hat, minimiert wird. Das Modell identifiziert einen Konflikt zwischen einer schnellen Initiierung der Origins und einer effizienten Verhinderung der DNA Rereplikation. Modellanalysen deuten darauf hin, dass eine zeitlich verzögerte Origininitiierung verursacht durch die multiple Phosphorylierung der Proteine Sic1 und Sld2 durch Cyclin-abhängige Kinasen, G1-Cdk bzw. S-Cdk, essentiell für die Lösung dieses Konfliktes ist. Insbesondere verschafft die Mehrfach-Phosphorylierung von Sld2 durch S-Cdk eine zeitliche Verzögerung, die robust gegenüber Veränderungen in der S-Cdk Aktivierungskinetik ist und außerdem eine nahezu simultane Aktivierung der Origins ermöglicht. Die berechnete Verteilung der Fertigstellungszeiten der RKs, oder die Verteilung der Originaktivierungszeiten, wird auch genutzt, um die Konsequenzen bestimmter Mutationen im Assemblierungsprozess auf das Kopieren des genetischen Materials in der S Phase des Zellzyklus zu simulieren., Before a cell divides it has to duplicate its entire genetic material. Eukaryotic genomes are replicated from multiple replication origins across the genome. This work is focused on the quantitative analysis of the underlying molecular mechanism that allows these origins to initiate DNA replication almost simultaneously and exactly once per cell cycle. Based on a vast amount of experimental findings, a molecular regulatory network is constructed that describes the assembly of the molecules at the replication origins that finally form complete replication complexes. Using mass–action kinetics, the molecular reactions are translated into a system of differential equations. To parameterize the mathematical model, the initial protein concentrations are taken from experimental data, while kinetic parameter sets are determined using an optimization approach, in particular a minimization of the duration, in which a minimum number of replication complexes has formed. The model identifies a conflict between the rapid initiation of replication origins and the efficient inhibition of DNA rereplication. Analyses of the model suggest that a time delay before the initiation of DNA replication provided by the multiple phosphorylations of the proteins Sic1 and Sld2 by cyclin-dependent kinases in G1 and S phase, G1-Cdk and S-Cdk, respectively, may be essential to solve this conflict. In particular, multisite phosphorylation of Sld2 by S-Cdk creates a time delay that is robust to changes in the S-Cdk activation kinetics and additionally allows the near-simultaneous activation of multiple replication origins. The calculated distribution of the assembly times of replication complexes, that is also the distribution of origin activation times, is then used to simulate the consequences of certain mutations in the assembly process on the copying of the genetic material in S phase of the cell cycle.

Apoptosis is a programmed cell death that plays a physiological role during development of the nervous system, while it displays a critical pathological function in chronic neurodegenerative diseases and acute neurological disorders, such as ischemic stroke, epilepsy and brain injury. This review aims to give an overview of the essential molecular pathways underlying neuroapoptosis and the complex intracellular and inter-cellular cross-talks which involve a large number of metabolic and signaling cascades that may also act synergistically during brain damage. Starting from accumulation of damaged/misfolded proteins that mainly characterizes neurodegenerative diseases, such as AD, PD and HD, we then consider the role of mitochondrial dysfunction, alterations of intracellular Ca2+ homeostasis and production of oxidative stress in the induction of neuronal apoptosis. The interplay between neuronal and glial cells is also discussed as a mechanism involved in most neurological conditions including acute (stroke and brain injury) and chronic neurodegenerations (ALS, MS and PNP). Since neuronal-glial network has a role in synaptic plasticity and glutamate metabolism, reactive gliosis may potentiate neuronal death by stimulating glutamate toxicity and disrupting neuronal connections. Finally, we present new insights on molecular mechanisms of neuroprotection, that suggest a new therapeutic strategy for the treatment of several neurological disorders

The insulin-like growth factor receptor I (IGF-IR) plays an essential role in transformation by promoting cell growth and protecting cancer cells from apoptosis. Aberrant IGF-IR signaling is implicated in several types of tumors, including carcinomas of the lung, breast, prostate, pancreas, liver, and colon. However, the contribution of the IGF-IR to the development of the transformed phenotype in urothelial cells has not been clearly established. In this study we demonstrated that the IGF-IR is overexpressed in invasive bladder cancer tissues compared with nonmalignant controls. We have investigated the role of the IGF-IR in bladder cancer by using urothelial carcinoma-derived 5637 and T24 cells. Although activation of the IGF-IR did not appreciably affect their growth, it did promote migration and stimulate in vitro wound closure and invasion. These effects required the activation of the Akt and Mitogen-activated protein kinase (MAPK) pathways as well as IGF-I-induced Akt- and MAPK-dependent phosphorylation of paxillin, which relocated at dynamic focal adhesions and was necessary for promoting motility in bladder cancer cells. Our results provide the first evidence for a role of the IGF-IR in motility and invasion of bladder cancer cells and support the hypothesis that the IGF-IR may play a critical role in the establishment of the invasive phenotype in urothelial neoplasia. Thus, the IGF-IR may also serve as a novel biomarker for bladder cancer. Copyright © American Society for Investigative Pathology.

Large-scale "omics" data are often represented as networks of interacting components, but such representation is inherently static and, as such, cannot provide a realistic picture of the temporal dynamics of complex cellular functions. These difficulties suggest moving to a modeling strategy that explicitly takes into account both the wiring of the components and the task they perform. From an engineering perspective, this problem resembles that of "circuit analysis". In this paper, we focus on a limited but relevant biological circuit, the G1 to S transition in yeast cell cycle, and investigate both the network representation and the corresponding circuit described by a mathematical model, by means of a wide range of numerical simulation analysis. Reliable predictions of system-level properties are achieved and the parameters that mostly affect these properties are found out. © 2010 Elsevier Inc. All rights reserved.

Eukaryotic genomes are duplicated from multiple replication origins exactly once per cell cycle. In Saccharomyces cerevisiae, a complex molecular network has been identified that governs the assembly of the replication machinery. Here we develop a mathematical model that links the dynamics of this network to its performance in terms of rate and coherence of origin activation events, number of activated origins, the resulting distribution of replicon sizes and robustness against DNA rereplication. To parameterize the model, we use measured protein expression data and systematically generate kinetic parameter sets by optimizing the coherence of origin firing. While randomly parameterized networks yield unrealistically slow kinetics of replication initiation, networks with optimized parameters account for the experimentally observed distribution of origin firing times. Efficient inhibition of DNA rereplication emerges as a constraint that limits the rate at which replication can be initiated. In addition to the separation between origin licensing and firing, a time delay between the activation of S phase cyclin-dependent kinase (S-Cdk) and the initiation of DNA replication is required for preventing rereplication. Our analysis suggests that distributive multisite phosphorylation of the S-Cdk targets Sld2 and Sld3 can generate both a robust time delay and contribute to switch-like, coherent activation of replication origins. The proposed catalytic function of the complex formed by Dpb11, Sld3 and Sld2 strongly enhances coherence and robustness of origin firing. The model rationalizes how experimentally observed inefficient replication from fewer origins is caused by premature activation of S-Cdk, while premature activity of the S-Cdk targets Sld2 and Sld3 results in DNA rereplication. Thus the model demonstrates how kinetic deregulation of the molecular network governing DNA replication may result in genomic instability

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in expo- nential growing batch cultures at faster growth rate, while localization of catalytic and regulatory sub- units is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol- grown cells appear to depend on both increase in molecule number and kcat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism

Molecular systems biology aims to describe the functions of complex biological processes through recursive integration of molecular analysis, modeling, simulation and theory. It focuses on networks that originate from interconnection of genes, proteins and metabolites whose dynamic interactions generate, as an emergent property of the system, the corresponding function. Although evolutionary optimized, intracellular biochemical parameters, such as the expression level of gene products or the affinity between two or more proteins, must have a permissible range that gives robustness against perturbations to the system. Using the yeast G1-to-S transition network as an example we show that sophisticated relations exist among network structure, emergent property and robustness. Different emergent properties are generated from the same network by changing the strength of its interactions, not only by altering expression level, but also through mono and multi-site phosphorylation/dephosphorylation. Besides, multi-site protein phosphorylation modules, widespread in cell cycle, may ensure robust and coherent timing of cell cycle transitions as it happens for the onset of DNA replication. In conclusion, the modulation of biological function/emergent property by modifying interaction strength provides an efficient, highly tunable device to regulate biological processes. Furthermore, the principles outlined herein may provide new insight to network analysis in drug discovery. © 2009 Elsevier B.V. All rights reserved.

Many cancer cells are characterized by high rate of glycolysis and reduced rate of aerobic respiration, whose mechanism is still elusive. Here we investigate the down-regulation of oxidative phosphorylation (OXPHOS) in K-ras transformed mouse fibroblasts as compared to a control counterpart. Transcriptional analysis showed different expression levels of several OXPHOS nuclear genes in the two cell lines. In particular, during the exponential growth phase most genes encoding proteins of Complex I were expressed at lower levels in transformed cells. Consistently, a significant decrease of Complex I content was found in transformed cells. Moreover, analysis of NAD-dependent respiration and ATP synthesis indicated a strong decrease of Complex I activity in the mitochondria from neoplastic cells, that was confirmed by direct assay of the enzyme redox activity. At variance, succinate-dependent respiration and ATP synthesis were not significantly affected. Taken together, our results provide the new insight that the reduction of respiration observed in K-ras transformed cells is specifically due to a Complex I activity decrease.

BACKGROUND:The integration of data from multiple genome-wide assays is essential for understanding dynamic spatio-temporal interactions within cells. Such integration, which leads to a more complete view of cellular processes, offers the opportunity to rationalize better the high amount of "omics" data freely available in several public databases.In particular, integration of microarray-derived transcriptome data with other high-throughput analyses (genomic and mutational analysis, promoter analysis) may allow us to unravel transcriptional regulatory networks under a variety of physio-pathological situations, such as the alteration in the cross-talk between signal transduction pathways in transformed cells. RESULTS:Here we sequentially apply web-based and statistical tools to a case study: the role of oncogenic activation of different signal transduction pathways in the transcriptional regulation of genes encoding proteins involved in the cAMP-PKA pathway. To this end, we first re-analyzed available genome-wide expression data for genes encoding proteins of the downstream branch of the PKA pathway in normal tissues and human tumor cell lines. Then, in order to identify mutation-dependent transcriptional signatures, we classified cancer cells as a function of their mutational state. The results of such procedure were used as a starting point to analyze the structure of PKA pathway-encoding genes promoters, leading to identification of specific combinations of transcription factor binding sites, which are neatly consistent with available experimental data and help to clarify the relation between gene expression, transcriptional factors and oncogenes in our case study. CONCLUSIONS:Genome-wide, large-scale "omics" experimental technologies give different, complementary perspectives on the structure and regulatory properties of complex systems. Even the relatively simple, integrated workflow presented here offers opportunities not only for filtering data noise intrinsic

BACKGROUND: The cell cycle is a complex process that allows eukaryotic cells to replicate chromosomal DNA and partition it into two daughter cells. A relevant regulatory step is in the G0/G1 phase, a point called the restriction (R) point where intracellular and extracellular signals are monitored and integrated.Subcellular localization of cell cycle proteins is increasingly recognized as a major factor that regulates cell cycle transitions. Nevertheless, current mathematical models of the G1/S networks of mammalian cells do not consider this aspect. Hence, there is a need for a computational model that incorporates this regulatory aspect that has a relevant role in cancer, since altered localization of key cell cycle players, notably of inhibitors of cyclin-dependent kinases, has been reported to occur in neoplastic cells and to be linked to cancer aggressiveness. RESULTS: The network of the model components involved in the G1 to S transition process was identified through a literature and web-based data mining and the corresponding wiring diagram of the G1 to S transition drawn with Cell Designer notation. The model has been implemented in Mathematica using Ordinary Differential Equations. Time-courses of level and of sub-cellular localization of key cell cycle players in mouse fibroblasts re-entering the cell cycle after serum starvation/re-feeding have been used to constrain network design and parameter determination. The model allows to recapitulate events from growth factor stimulation to the onset of S phase. The R point estimated by simulation is consistent with the R point experimentally determined. CONCLUSION: The major element of novelty of our model of the G1 to S transition is the explicit modeling of cytoplasmic/nuclear shuttling of cyclins, cyclin-dependent kinases, their inhibitor and complexes. Sensitivity analysis of the network performance newly reveals that the biological effect brought about by Cki overexpression is strictly dependent on whether

Background: Oncogene activation plays a role in metabolic reprogramming of cancer cells. We have previously shown that K-ras transformed fibroblasts have a stronger dependence on glycolysis and a reduced oxidative phosphorylation ability as compared to their normal counterparts. Another metabolic adaptation of cancer cells, that has long been established, is their propensity to exhibit increased glutamine consumption, although the effects induced by glutamine deprivation on cancer cells are still controversial. Methodology and Principal Findings: Here, by using nutritional perturbations and molecular physiology, we show that reduction or complete depletion of glutamine availability in K-ras transformed fibroblasts causes a strong decrease of proliferation ability and a slower re-entry of synchronized cells into the cell cycle. The reduced proliferation is accompanied by sustained expression of cyclin D and E, abortive S phase entrance and is dependent on Ras signalling deregulation, since it is rescued by expression of a dominant negative guanine nucleotide exchange factor. The growth potential of transformed cells as well as the ability to execute the G1 to S transition is restored by adding the four deoxyribonucleotides, indicating that the arrest of proliferation of K-ras transformed cells induced by glutamine depletion is largely due to a reduced supply of DNA in the presence of signalling pathways promoting G1 to S transition. Conclusions and Significance: Our results suggest that the differential effects of glutamine and glucose on cell viability are not a property of the transformed phenotype per se, but rather depend on the specific pathway being activated in transformation. For instance, myc-overexpressing cells have been reported to die under glutamine depletion and not under glucose shortage, while the opposite holds for ras-transformed fibroblasts as shown in this paper. These different responses of transformed cells to nutritional stress should be taken

Intrinsically disordered proteins (IDPs) carry out important biological functions and offer an instructive model system for folding and binding studies. However, their structural characterization in the absence of interactors is hindered by their highly dynamic conformation. The cyclin-dependent-kinase inhibitor (Cki) Sic1 from Saccharomyces cerevisiae is a key regulator of the yeast cell cycle, which controls entrance into S phase and coordination between cell growth and proliferation. Its last 70 out of 284 residues display functional and structural homology to the inhibitory domain of mammalian p21 and p27. Sic1 has escaped systematic structural characterization until now. Here, complementary biophysical methods are applied to the study of conformational properties of pure Sic1 in solution. Based on sequence analysis, gel filtration, circular dichroism (CD), electrospray-ionization mass spectrometry (ESI-MS), and limited proteolysis, it can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an IDP. However, the results of these experiments indicate, at the same time, that the protein displays some content in secondary and tertiary structure, having properties similar to those of molten globules or premolten globules. Proteolysis-hypersensitive sites cluster at the N-terminus and in the middle of the molecule, whereas the most structured region resides at the C-terminus, including part of the inhibitory domain and the casein-kinase-2 (CK2) phosphorylation target S201. The mutations S201A and S201E, which are known to affect Sic1 function, do not have significant effects on the conformational properties of the pure protein. Proteins 2009;76:731-746. (c) 2009 Wiley-Liss, Inc.

Model organisms and in particular the budding yeast Saccharomyces cerevisiae have been instrumental in advancing our understanding of cell cycle progression. The asymmetric division of the budding yeast and the tight coupling between cell growth and division have challenged the theoretical understanding of the cell size structure of growing yeast populations. Past efforts have centered on modeling the steady-state theoretical age distribution for asymmetric division from which a cell size distribution can be derived assuming dispersion of cell size within each age class. Different developments, especially in the field of flow cytometry, allowed the determination of a number of cellular properties and their joint distributions for the entire population and the different subpopulations as well. A new rigorous framework for modeling directly the dynamics of size distributions of structured yeast populations has been proposed, which readily extends to modeling of more complex conditions, such as transient growth. Literature on the structure of growing yeast populations and modeling of cell cycle progression is reviewed. (C) 2008 international Society for Advancement of Cytometry, Model organisms and in particular the budding yeast Saccharomyces cerevisiae have been instrumental in advancing our understanding of cell cycle progression. The asymmetric division of the budding yeast and the tight coupling between cell growth and division has challenged the theoretical understanding of the cell size structure of growing yeast populations. Past efforts have centred on modelling the steady-state theoretical age distribution for asymmetric division from which a cell size distribution can be derived assuming dispersion of cell size within each age class. Recent developments, especially in the field of flow cytometry, allowed the determination of a number of cellular properties and their joint distributions for both the entire population and the different subpopulations as well. A new rigorous framework for modelling directly the dynamics of size distributions of structured yeast populations has been recently proposed, which readily extends to modelling of more complex conditions, such as transient growth. Literature on the structure of growing yeast populations and modelling of cell cycle progression is reviewed.

Following a brief description of the operational procedures of systems biology (SB), the cell cycle of budding yeast is discussed as a successful example of a top-down SB analysis. After the reconstruction of the steps that have led to the identification of a sizer plus timer network in the G1 to S transition, it is shown that basic functions of the cell cycle (the setting of the critical cell size and the accuracy of DNA replication) are systemlevel properties, detected only by integrating molecular analysis with modelling and simulation of their underlying networks. A detailed network structure of a second relevant regulatory step of the cell cycle, the exit from mitosis, derived from extensive data mining, is constructed and discussed. To reach a quantitative understanding of how nutrients control, through signalling, metabolism and transcription, cell growth and cycle is a very relevant aim of SB. Since we know that about 900 gene products are required for cell cycle execution and control in budding yeast, it is quite clear that a purely systematic approach would require too much time. Therefore lines for a modular SB approach, which prioritises molecular and computational investigations for faster cell cycle understanding, are proposed. The relevance of the insight coming from the cell cycle SB studies in developing a new framework for tackling very complex biological processes, such as cancer and aging, is discussed.

Yeast cells undergoing a nutritional shift-up from a poor to a rich carbon source take several hours to adapt to the novel, richer carbon source. The budding index is a physiologically relevant "global" parameter that reflects the complex links between cell growth and division that are both coordinately and deeply affected by nutritional conditions. We used changes in budding index as a guide to choose appropriate, relevant time points during an ethanol to glucose nutritional shift-up for preparation of samples for the analysis of proteome by two-dimensional electrophoresis/mass spectrometry. About 600 spots were detected. 90 spots, mostly comprising proteins involved in intermediary metabolism, protein synthesis, and response to stress, showed differential expression after glucose addition. Among modulated proteins we identified a protein of previously unknown function, Gvp36, showing a transitory increase corresponding to the drop of the fraction of budded cells. A gvp36Delta strain shares several phenotypes (including general growth defects, heat shock, and high salt sensitivity, defects in polarization of the actin cytoskeleton, in endocytosis and in vacuolar biogenesis, defects in entering stationary phase upon nutrient starvation) with secretory pathway mutants and with mutants in genes encoding the two previously known yeast BAR proteins (RSV161 and RSV167). We thus propose that Gvp36 represents a novel yeast BAR protein involved in vesicular traffic and in nutritional adaptation.

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 °C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 °C and a shift of 10 °C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29¿42 °C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Protein kinase CK2 is a heterotetramer composed of two catalytic and two regulatory subunits. In Saccharomyces cerevisiae the catalytic subunits (¿ and ¿¿) are encoded by the CKA1, CKA2 genes. cka1¿cka2ts mutants arrest cell cycle in both G1 and G2/M at 37°C. Hence, it has been proposed that CK2 plays an important role in cell cycle progression and several cell cycle proteins have been reported to be CK2 substrates. We have previously shown that Sic1, the inhibitor of Clb5-Cdc28 complexes required for the G1/S transition, is a physiologically relevant CK2 substrate. Here we show that CK2 inactivation up-regulates Sic1 level resulting in severe down-regulation of Clb5-Cdc28 kinase activity. Concurrent inactivation of Sic1 and CK2 leads to accumulation of cells with a post-synthetic DNA content and short/elongated spindles, typical of cells arrested in mitosis. These findings indicate that Sic1 plays a major role during G1 arrest of CK2-inactivated cells

The rapamycin-sensitive (TOR) signalling pathway in Saccharomyces cerevisiae controls growth and cell proliferation in response to nutrient availability. Rapamycin treatment causes cells to arrest growth in G1 phase. The mechanism by which the inhibition of the TOR pathway regulates cell cycle progression is not completely understood. Here we show that rapamycin causes G1 arrest by a dual mechanism that comprises downregulation of the G1-cyclins Cln1-3 and upregulation of the Cdk inhibitor protein Sic1. The increase of Sic1 level is mostly independent of the downregulation of the G1 cyclins, being unaffected by ectopic CLN2 expression, but requires Sic1 phosphorylation of Thr173, because it is lost in cells expressing Sic1(T173A). Rapamycin-mediated Sic1 upregulation involves nuclear accumulation of a more stable, non-ubiquitinated protein. Either SIC1 deletion or CLN3 overexpression results in non-cell-cycle-specific arrest upon rapamycin treatment and makes cells sensitive to a sublethal dose of rapamycin and to nutrient starvation. In conclusion, our data indicate that Sic1 is involved in rapamycin-induced G1 arrest and that deregulated entrance into S phase severely decreases the ability of a cell to cope with starvation conditions induced by nutrient depletion or which are mimicked by rapamycin treatment.

Early reports on the production by both bacterial and eukaryotic cells of enzymes able to degrade lipid substrates date to over a century ago. Since then, research on lipolytic enzymes that includes lipases, esterases, phospholipases has been driven by their central roles in lipid metabolism and in signal transduction. Lipases are generally versatile enzymes that accept a broad range of substrates (i.e. aliphatic, alicyclic, bicyclic and aromatic esters, thioesters, activated amines) whilst maintaining high regio-, chemo- and enantioselectivity. The stability of most lipases in organic solvents paves the way for their exploitation in organic synthesis: in esterification, transesterification, aminolysis and oximolysis reactions (Drauz and Waldman, 1995). Such properties make lipases key players in the industrial enzyme sector (Schmid and Verger, 1998; Bornscheuer, 2000; Kirk et al., 2002; Jaeger and Eggert, 2002; Gupta et al., 2004). In this chapter we review the fundamental knowledge available on lipases, with particular emphasis on the relationship between the sequence, structure and function of those most commonly used in industrial processes. On the basis of this knowledge, novel and improved lipases may be generated, able to meet the requirements for robustness, selectivity and catalytic performances posed by modern biocatalysis. © 2007 Springer.

The eukaryotic cell cycle is the repeated sequence of events that enable the division of a cell into two daughter cells. It is divided into four phases: G1, S, G2, and M. Passage through the cell cycle is strictly regulated by a molecular interaction network, which involves the periodic synthesis and destruction of cyclins that bind and activate cyclin-dependent kinases that are present in nonlimiting amounts. Cyclin-dependent kinase inhibitors contribute to cell cycle control. Budding yeast is an established model organism for cell cycle studies, and several mathematical models have been proposed for its cell cycle. An area of major relevance in cell cycle control is the G1 to S transition. In any given growth condition, it is characterized by the requirement of a specific, critical cell size, PS, to enter S phase. The molecular basis of this control is still under discussion. The authors report a mathematical model of the G1 to S network that newly takes into account nucleo/cytoplasmic localization, the role of the cyclin-dependent kinase Sic1 in facilitating nuclear import of its cognate Cdk1-Clb5, Whi5 control, and carbon source regulation of Sic1 and Sic1-containing complexes. The model was implemented by a set of ordinary differential equations that describe the temporal change of the concentration of the involved proteins and protein complexes. The model was tested by simulation in several genetic and nutritional setups and was found to be neatly consistent with experimental data. To estimate PS, the authors developed a hybrid model including a probabilistic component for firing of DNA replication origins. Sensitivity analysis of PS provides a novel relevant conclusion: PS is an emergent property of the G 1 to S network that strongly depends on growth rate. © 2007 Barberis et al.

Neurotrophins are structurally related proteins regulating brain development and function. Molecular evolution studies of neurotrophins and their receptors are essential for understanding the mechanisms underlying the coevolution processes of these gene families and how they correlate with the increased complexity of the vertebrate nervous system. In order to improve our current knowledge of the molecular evolution of neurotrophins and receptors, we have collected all information available in the literature and analyzed the genome database for each of them. Statistical analysis of aminoacid and nucleotide sequences of the neurotrophin and Trk family genes was applied to both complete genes and mature sequences, and different phylogenetic methods were used to compare aminoacid and nucleotide sequences variability among the different species. All collected data favor a model in which several rounds of genome duplications might have facilitated the generation of the many different neurotrophins and the acquisition of specific different functions correlated with the increased complexity of the vertebrate nervous system during evolution.We report findings that refine the structure of the evolutionary trees for neurotrophins and Trk receptors families, indicate different rates of evolution for eachmember of the two families, and newly demonstrate that the NGF-like genes found in Fowlpox and Canarypox viruses are closely related to reptile NGF.