Cargo trafficking between endosomes and the trans-Golgi network

The retrograde membrane transport pathways from endosomes to the trans-Golgi network (TGN) are now recognized as critical intracellular pathways to recycle and shuttle a selective subgroup of membrane proteins, including sorting receptors, membrane-bound enzymes, transporters, as well as providing an avenue for the intracellular transport of various bacterial toxins. Multiple pathways from endosomes to the TGN have now been defined which differ between the cargo transported and the machinery used. Here, we review advances in these pathways and the requirement for TGN organization, and also discuss the development of unbiased analytical approaches to quantitatively track cargo that use these endosome-to-TGN pathways.     

Connections between the globus pallidus and putamen and the hypothalamus and subthalamus

In 16 adult cats with electrolytically destructed external and internal parts of the globus pallidus and in 8 cats with destructed putamen direct strio-pallido-hypothalamic and strio-pallido-subthalamic pathways have been studied. Degeneration of the axonal preterminals and terminals have been examined in preparations treated after Nauta--Gygax, Nauta--Laidlow, Finck--Heimer with simultaneous additional staining of the nuclei with cresyl violet after Kawamura--Niimi. Direct pallido- and putamen-hypothalamic pathways to nuclei of the grey tubercle, posterior and lateral nuclei of the hypothalamus were stated. Direct pathways from the putamen to the subthalamic nucleus have been revealed, however, these pathways are represented in less degree than those of pallido-subthalamic connections. Direct pathways from the external portion of the globus pallidus and putamen to the subthalamic nucleus are more pronounced and represented by greater numbers of projections than those of strio-pallido-hypothalamic origin.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Exploring the diversity of complex metabolic networks

MOTIVATION: Metabolism, the network of chemical reactions that make life possible, is one of the most complex processes in nature. We describe here the development of a computational approach for the identification of every possible biochemical reaction from a given set of enzyme reaction rules that allows the de novo synthesis of metabolic pathways composed of these reactions, and the evaluation of these novel pathways with respect to their thermodynamic properties. RESULTS: We applied this framework to the analysis of the aromatic amino acid pathways and discovered almost 75,000 novel biochemical routes from chorismate to phenylalanine, more than 350,000 from chorismate to tyrosine, but only 13 from chorismate to tryptophan. Thermodynamic analysis of these pathways suggests that the native pathways are thermodynamically more favorable than the alternative possible pathways. The pathways generated involve compounds that exist in biological databases, as well as compounds that exist in chemical databases and novel compounds, suggesting novel biochemical routes for these compounds and the existence of biochemical compounds that remain to be discovered or synthesized through enzyme and pathway engineering. AVAILABILITY: Framework will be available via web interface at http://systemsbiology.northwestern.edu/BNICE (site under construction). CONTACT: vassily@northwestern.edu or broadbelt@northwestern.edu SUPPLEMENTARY INFORMATION: http://systemsbiology.northwestern.edu/BNICE/publications.     

Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway.

In addition to the CDP-choline pathway for phosphatidylcholine (PC) synthesis, the liver has a unique phosphatidylethanolamine (PE) methyltransferase activity for PC synthesis via three methylations of the ethanolamine moiety of PE. Previous studies indicate that the two pathways are functionally different and not interchangeable even though PC is the common product of both pathways. This study was designed to test the hypothesis that these two pathways produce different profiles of PC species. The PC species from these two pathways were labeled with specific stable isotope precursors, D9-choline and D4-ethanolamine, and analyzed by electrospray tandem mass spectrometry. Our studies revealed a profound distinction in PC profiles between the CDP-choline pathway and the PE methylation pathway. PC molecules produced from the CDP-choline pathway were mainly comprised of medium chain, saturated (e.g. 16:0/18:0) species. On the other hand, PC molecules from the PE methylation pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g. 18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver.     

Genomics and expression profiles of the Hedgehog and Notch signaling pathways in sea urchin development

The Hedgehog (Hh) and Notch signal transduction pathways control a variety of developmental processes including cell fate choice, differentiation, proliferation, patterning and boundary formation. Because many components of these pathways are conserved, it was predicted and confirmed that pathway components are largely intact in the sea urchin genome. Spatial and temporal location of these pathways in the embryo, and their function in development offer added insight into their mechanistic contributions. Accordingly, all major components of both pathways were identified and annotated in the sea urchin Strongylocentrotus purpuratus genome and the embryonic expression of key components was explored. Relationships of the pathway components, and modifiers predicted from the annotation of S. purpuratus, were compared against cnidarians, arthropods, urochordates, and vertebrates. These analyses support the prediction that the pathways are highly conserved through metazoan evolution. Further, the location of these two pathways appears to be conserved among deuterostomes, and in the case of Notch at least, display similar capacities in endomesoderm gene regulatory networks. RNA expression profiles by quantitative PCR and RNA in situ hybridization reveal that Hedgehog is produced by the endoderm beginning just prior to invagination, and signals to the secondary mesenchyme-derived tissues at least until the pluteus larva stage. RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm. Functional analyses in future studies will embed these pathways into the growing knowledge of gene regulatory networks that govern early specification and morphogenesis.     

Discovery and analysis of novel metabolic pathways for the biosynthesis of industrial chemicals: 3‐hydroxypropanoate

Sustainable microbial production of high‐value organic compounds such as 3‐hydroxypropanoate (3HP) is becoming an increasingly attractive alternative to organic syntheses that utilize petrochemical feedstocks. We applied the Biochemical Network Integrated Computational Explorer (BNICE) framework to the automated design and evaluation of novel biosynthetic routes for the production of 3HP from pyruvate. Among the pathways generated by the BNICE framework were all of the known pathways for the production of 3HP as well as numerous novel pathways. The pathways generated by BNICE were ranked based on four criteria: pathway length, thermodynamic feasibility, maximum achievable yield to 3HP from glucose, and maximum achievable activity at which 3HP can be produced. Four pathways emerged from this ranking as the most promising for the biosynthesis of 3HP, and three of these pathways, including the shortest pathways discovered, were novel. We also discovered novel routes for the biosynthesis of 28 commercially available compounds that are currently produced exclusively through organic synthesis. Examination of the optimal pathways for the biosynthesis of these 28 compounds in E. coli revealed pyruvate and succinate to be ideal intermediates for achieving high product yields from glucose. Biotechnol. Bioeng. 2010; 106: 462–473. © 2010 Wiley Periodicals, Inc.     

The development of the early atrioventricular conduction system in the embryonic heart

Recent electrophysiological evidence indicates that periodic spontaneous depolarizations occur in the primordial heart of the bird (and presumably mammal) even before the myocardial cells can contract, and these are initiated in the primordial sinoatrial region. As contractions are generated, these then establish a peristaltic wave. From that time on, during ontogenesis, the contractile sequence follows a regular pattern of development. As chambers form they contract sequentially in the direction of blood flow, even though, in the twisted configuration, myocardial continuities suggest the possibility of short-circuiting the electrical conduction pathways from atrium to bulbus. This implies that, even at these early stages, the electrical properties of the myocardium are not isotropic, and that specialized conduction pathways must exist. To the present time, electrophysiological techniques have limited the direct evidence that can be obtained on these delicate electrically specialized pathways. However, microscopical techniques have permitted studies on the morphological development of the tissue and of the cells in the various regions of the myocardium. The present paper traces the development of cell morphology in these regions, including the development of structural nodes and proximal ventricular fibre pathways, and from these observations, the manner in which the electrical conduction pathways are believed to develop is suggested.     

Retrograde trafficking and plasma membrane recycling pathways of the budding yeast Saccharomyces cerevisiae

The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome‐to‐Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.     

Adenine and adenosine salvage pathways in erythrocytes and the role of S-adenosylhomocysteine hydrolase. A theoretical study using elementary flux modes

This article is devoted to the study of redundancy and yield of salvage pathways in human erythrocytes. These cells are not able to synthesize ATP de novo. However, the salvage (recycling) of certain nucleosides or bases to give nucleotide triphosphates is operative. As the salvage pathways use enzymes consuming ATP as well as enzymes producing ATP, it is not easy to see whether a net synthesis of ATP is possible. As for pathways using adenosine, a straightforward assumption is that these pathways start with adenosine kinase. However, a pathway bypassing this enzyme and using S-adenosylhomocysteine hydrolase instead was reported. So far, this route has not been analysed in detail. Using the concept of elementary flux modes, we investigate theoretically which salvage pathways exist in erythrocytes, which enzymes belong to each of these and what relative fluxes these enzymes carry. Here, we compute the net overall stoichiometry of ATP build-up from the recycled substrates and show that the network has considerable redundancy. For example, four different pathways of adenine salvage and 12 different pathways of adenosine salvage are obtained. They give different ATP/glucose yields, the highest being 3:10 for adenine salvage and 2:3 for adenosine salvage provided that adenosine is not used as an energy source. Implications for enzyme deficiencies are discussed.     

Defining the limits: Protein aggregation and toxicity in vivo

Abstract

The proper folding of proteins to their functional forms is essential to cellular homeostasis. Perhaps not surprisingly, cells have evolved multiple pathways, some overlapping and others complementary, to resolve mis-folded proteins when they arise, ranging from refolding through the action of molecular chaperones to elimination through regulated proteolytic mechanisms. These protein quality control pathways are sufficient, under normal conditions, to maintain a functioning proteome, but in response to diverse environmental, genetic and/or stochastic events, protein mis-folding exceeds the corrective capacity of these pathways, leading to the accumulation of aggregates and ultimately toxicity. Particularly devastating examples of these effects include certain neurodegenerative diseases, such as Huntington’s Disease, which are associated with the expansion of polyglutamine tracks in proteins. In these cases, protein mis-folding and aggregation are clear contributors to pathogenesis, but uncovering the precise mechanistic links between the two events remains an area of active research. Studies in the yeast Saccharomyces cerevisiae and other model systems have uncovered previously unanticipated complexity in aggregation pathways, the contributions of protein quality control processes to them and the cellular perturbations that result from them. Together these studies suggest that aggregate interactions and localization, rather than their size, are the crucial considerations in understanding the molecular basis of toxicity.     

Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways

A growing body of evidence indicates that phenylpropanoid and flavonoid metabolism is catalyzed, not by free-floating 'soluble' enzymes, but via one or more membrane-associated multienzyme complexes. This type of macromolecular organization has important implications for the overall efficiency, specificity, and regulation of these pathways. Classical biochemical studies of phenylpropanoid and flavonoid metabolism have laid a solid foundation for this model, providing evidence of the channeling of intermediates between enzyme active sites and co-localization of enzymes in cell membranes. This work is now being extended using transgenic plants to determine how the partitioning of metabolites within these pathways is controlled, as well as applying sensitive methods to define specific interactions among the individual enzymes. Information from these studies promises to provide new insights into the structuring of biosynthetic pathways within cells, which should lead to more effective means for engineering the production of plant metabolites with nutritional and agronomic importance.     

Hormonal signaling and signal pathway crosstalk in the control of myometrial calcium dynamics

Understanding the basis for the control of myometrial contractant and relaxant signaling pathways is important to understanding how to manage myometrial contractions. Signaling pathways are influenced by the level of expression of the signals and signal pathway components, the location of these components in the appropriate subcellular environment, and covalent modification. Crosstalk between these pathways regulates the effectiveness of signal transduction and represents an important way by which hormones can regulate phenotype. This review deals primarily with signaling pathways that control Ca2+ entry and intracellular release, as well as the interplay between these pathways.     

Activities of the protein kinases STK, PI3K, MEK, and ERK are required for the development of the head organizer in Hydra magnipapillata

The development of the hydra's head and its hypostome has been studied at the molecular level. Many genes have been cloned from hydra as potential candidates that control the development of its head. Much work was performed on the mechanisms controlling expression of these genes in the position-dependent manner. Moreover, there have been data to support the involvement of three main signaling pathways that involve PKC, SRC, and PI3K kinases in the regulation of the head formation and in the expression of several head-specific genes. In this report, we present data supporting the participation of these three signaling pathways on the development of the hypostome. We used grafting experiments and inhibitors of the specific kinases to show the participation of these enzymes in hypostome formation. From our results, we postulate that these signal transduction pathways regulate the very early stages of the head development, most likely at the point when the cells start to differentiate to form the head organizer.     

Reconstruction and in silico analysis of the MAPK signaling pathways in the human blood fluke, Schistosoma japonicum

At present, little is known about signal transduction mechanisms in schistosomes, which cause the disease of schistosomiasis. The mitogen-activated protein kinase (MAPK) signaling pathways, which are evolutionarily conserved from yeast to Homo sapiens, play key roles in multiple cellular processes. Here, we reconstructed the hypothetical MAPK signaling pathways in Schistosoma japonicum and compared the schistosome pathways with those of model eukaryote species. We identified 60 homologous components in the S. japonciumMAPK signaling pathways. Among these, 27 were predicted to be full-length sequences. Phylogenetic analysis of these proteins confirmed the evolutionary conservation of the MAPK signaling pathways. Remarkably, we identified S. japonicum homologues of GTP-binding protein beta and alpha-I subunits in the yeast mating pathway, which might be involved in the regulation of different life stages and female sexual maturation processes as well in schistosomes. In addition, several pathway member genes, including ERK, JNK, Sja-DSP, MRAS and RAS, were determined through quantitative PCR analysis to be expressed in a stage-specific manner, with ERK, JNK and their inhibitor Sja-DSP markedly upregulated in adult female schistosomes.     

Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane-related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum-related transport pathways throughout eukaryotic evolution.     

p53 Regulation of the IGF-1/AKT/mTOR Pathways and the Endosomal Compartment

In response to various stress signals, which introduce infidelity into the processes of cell growth and division, p53 initiates cell-cycle arrest, apoptosis, or senescence to maintain fidelity throughout the cell cycle. Although these functions are traditionally thought of as the major functions of the p53 protein for tumor suppression, recent studies have revealed some additional novel functions of the p53 pathway. These include the down-regulation of two central cell-growth pathways, the IGF/AKT-1 and mTOR pathways, and the up-regulation of the activities of the endosomal compartment. The IGF-1/AKT and mTOR pathways are two evolutionarily conserved pathways that play critical roles in regulation of cell proliferation, survival, and energy metabolism. In response to stress, p53 transcribes a group of critical negative regulators in these two pathways, including IGF-BP3, PTEN, TSC2, AMPK β1, and Sestrin1/2, which leads to the reduction in the activities of these two pathways. Furthermore, p53 transcribes several critical genes regulating the endosomal compartment, including TSAP6, Chmp4C, Caveolin-1, and DRAM, and increases exosome secretion, the rate of endosomal removal of growth factor receptors (e.g., EGFR) from cell surface, and enhances autophagy. These activities all function to slow down cell growth and division, conserve and recycle cellular resources, communicate with adjacent cells and dendritic cells of the immune system, and inform other tissues of the stress signals. This coordinated regulation of IGF-1/AKT/mTOR pathways and the endosomal compartment by the p53 pathway integrates the molecular, cellular, and systemic levels of activities and prevents the accumulations of errors in response to stress and restores cellular homeostasis after stress.     

Signalling for survival and death in neurones: the role of stress-activated kinases, JNK and p38

The pathways involved in neuronal survival or death have been extensively studied mainly in cell lines. Recent evidence has suggested that activation of the stress activated pathways, jun N-terminal kinase (JNK) and p38 may play important roles in neuronal cell death or regeneration. In this review we will discuss these pahtways in detail. We will examine the evidence that these pathways are important in neuronal cell death. Finally we will review the evidence that inhibitors of these pathways have a neuroprotective effect both in vitro and in vivo.     

Central neural mechanisms that interrelate sensory and affective dimensions of pain

The perception of pain is highly complex, and requires neural integration from a variety of routes. Spinal pathways to the amygdala, hypothalamus, reticular formation, medial thalamic nuclei, and limbic cortical structures transmit information involved arousal, bodily regulation, and emotional responses. Other, albeit indirect, pathways can carry signals to these same structures, for example, from spinal pathways to somatosensory thalamic and cortical areas, and from these to cortical limbic structures. Indirect cortico-limbic pathways integrate nociception with information about the status of the body and indirect routes must culminate in the prioritization of emotions and responses to pain.