Mechanisms of signaling and related enzymes

Most enzymes involved in cell signaling, such as protein kinases, protein phosphatases, GTPases, and nucleotide cyclases catalyze nucleophilic substitutions at phosphorus. When possible, the mechanisms of such enzymes are most clearly described quantitatively in terms of how associative or dissociative they are. The mechanisms of cell signaling enzymes range from ≤8% associative (cAMP-dependent protein kinase) to ≈50% associative (G protein Giα1). Their catalytic powers range from 10^5.7 (p21^ras) to 10^11.7 (λ Ser-Thr protein phosphatase), usually comparable in magnitude with those of nonsignaling enzymes of the same mechanistic class. Exceptions are G proteins, which are 10³- to 10⁵-fold poorer catalysts than F1 and myosin ATPases. The lower catalytic powers of G proteins may be ascribed to the absence of general base catalysis, and additionally in the case of p21^ras, to the absence of a catalytic Arg residue, which interacts with the transition state. From kinetic studies of mutant and metal ion substituted enzymes, the catalytic powers of cell signaling and related enzymes can be rationalized quantitatively by factors contributed by metal ion catalysis (≥10⁵), general acid catalysis (≈10^3±1), general base catalysis (≈10^3±1), and transition-state stabilization by cationic and hydrogen bond donating residues (≈10^3±1). Proteins 29:401–416, 1997. © 1997 Wiley-Liss, Inc.     

Prolyl hydroxylase domain enzymes and their role in cell signaling and cancer metabolism

The prolyl hydroxylase domain (PHD) enzymes regulate the stability of the hypoxia-inducible factor (HIF) in response to oxygen availability. During oxygen limitation, the inhibition of PHD permits the stabilization of HIF, allowing the cellular adaptation to hypoxia. This adaptation is especially important for solid tumors, which are often exposed to a hypoxic environment. However, and despite their original role as the oxygen sensors of the cell, PHD are currently known to display HIF-independent and hydroxylase-independent functions in the control of different cellular pathways, including mTOR pathway, NF-kB pathway, apoptosis and cellular metabolism. In this review, we summarize the recent advances in the regulation and functions of PHD in cancer signaling and cell metabolism.     

The role of ectonucleotides metabolizing enzymes in purinergic signaling

Purinergic signaling plays an important role in the regulation of many physiological processes. The concentration of nucleotides in extracellular space is controlled by at least two families of nucleotidases: NPPases and NTPDases. These families are examples of convergent evolution of proteins. Above ezymes are not phylogenetically related, but they catalyze the same type of reaction. They hydrolyzed tri- and diphosphonucleosides to monophosphonucleosides and orthophosphate or pyrophosphate. This degradation terminates the nucleotide signaling process and also produces other signaling molecules like ADP, and with 5'-nucleotidase, adenosine. Most of known animal NPPases and NTPDases were found as membranous ectoenzymes or soluble proteins localized in tissue fluids. The aim of this work is to provide information about localization, structure, properties and function of NPPases and NTPDases in the regulation of extracellular concentration of nucleotides and purinergic signaling.     

The University of Minnesota Biocatalysis/Biodegradation Database: emphasizing enzymes

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.ahc.umn.edu/) provides curated information on microbial catabolic enzymes and their organization into metabolic pathways. Currently, it contains information on over 400 enzymes. In the last year the enzyme page was enhanced to contain more internal and external links; it also displays the different metabolic pathways in which each enzyme participates. In collaboration with the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology, 35 UM-BBD enzymes were assigned complete EC codes during 2000. Bacterial oxygenases are heavily represented in the UM-BBD; they are known to have broad substrate specificity. A compilation of known reactions of naphthalene and toluene dioxygenases were recently added to the UM-BBD; 73 and 108 were listed respectively. In 2000 the UM-BBD is mirrored by two prestigious groups: the European Bioinformatics Institute and KEGG (the Kyoto Encyclopedia of Genes and Genomes). Collaborations with other groups are being developed. The increased emphasis on UM-BBD enzymes is important for predicting novel metabolic pathways that might exist in nature or could be engineered. It also is important for current efforts in microbial genome annotation.     

Inactivation by UDP-glucuronosyltransferase enzymes: the end of androgen signaling

Conjugation by UDP-Glucuronosyltransferase (UGT) is the major pathway of androgen metabolism and elimination in the human. High concentrations of glucuronide conjugates of androsterone (ADT) and androstane-3alpha,17beta-diol (3alpha-diol) are present in circulation and several studies over the last 30 years have concluded that the serum levels of these metabolites might reflect the androgen metabolism in several tissues, including the liver and androgen target tissues. Three UGT2B enzymes are responsible for the conjugation of DHT and its metabolites ADT and 3alpha-diol: UGT2B7, B15 and B17. UGT2B7 is expressed in the liver and skin whereas UGT2B15 and B17 were found in the liver, prostate and skin. Very specific antibodies against each UGT2B enzyme have been obtained and used for immunohistochemical studies in the human prostate. It was shown that UGT2B17 is expressed in basal cells whereas UGT2B15 is only localized in luminal cells, where it inactivates DHT. By using LNCaP cells, we have also demonstrated that the expression and activity of UGT2B15 and B17 are modulated by several endogenous prostate factors including androgen. Finally, to study the physiological role of UGT2B enzymes, transgenic mice bearing the human UGT2B15 gene were recently obtained. A decrease in reproductive tissue weight from transgenic animals compared to those from control animals was observed. In conclusion, the conjugation by UGT2B7, B15 and B17, which represents a non-reversible step in androgen metabolism, is an important means by which androgens are regulated locally. It is also postulated that UGT enzymes protect the tissue from deleteriously high concentrations of active androgen.     

Modeling cell signaling networks

Cell signaling pathways interact with one another to form networks in mammalian systems. Such networks are complex in their organization and exhibit emergent properties such as bistability and ultrasensitivity. Analysis of signaling networks requires a combination of experimental and theoretical approaches including the development and analysis of models. This review focuses on theoretical approaches to understanding cell signaling networks. Using heterotrimeric G protein pathways an example, we demonstrate how interactions between two pathways can result in a network that contains a positive feedback loop and function as a switch. Different mathematical approaches that are currently used to model signaling networks are described, and future challenges including the need for databases as well as enhanced computing environments are discussed.     

Small-molecule inhibitors of cell signaling

Small-molecule inhibitors of several intracellular signaling proteins, mostly protein kinases, show tremendous selectivity and potency. The complexity and redundancy of signaling pathways presents opportunities for therapeutic selectivity and some clinical results are remarkable. New strategies are being developed to interfere with previously intractable targets, such as protein-protein interactions.     

Models of cell signaling pathways

Cellular signaling circuits handle an enormous range of computations. Beyond the housekeeping, replicating and other functions of individual cells, signaling circuits must implement the immensely complex logic of development and function of multicellular organisms. Computer models are useful tools to understand this complexity. Recent studies have extended such models to include electrical, mechanical and spatial details of signaling, and to address the stochastic effects that arise when small numbers of molecules interact. Increasing numbers of models have been developed in close conjunction with experiments, and this interplay gives a deeper and more reliable insight into signaling function.     

Guard cell signaling.

The plant hormone abscisic acid (ABA) regulates the aperture of the stomatal pore. The recent identification of new intermediates involved in ABA signaling suggests that this complex pathway is organized as a module-based network.     

Red cell enzymes of the Pongidae

Summary The red cell enzymes acid phosphatase, adenylate kinase, adenosine deaminase and phosphoglucomutase were analyzed by horizontal starch gel electrophoresis in 43 members of the family Pongidae: Pongo pygmaeus (n=10), Gorilla g. gorilla (n=8), Pan troglodytes (n=22) and Pan paniscus (n=3).In all the Pongidae a red cell acid phosphatase zymogram corresponding to the phenotype B in man was found. The adenylate kinase corresponded to the human phenotype AK 1. All the Pongidae showed the same homozygous adenosine deaminase phenotype which was different from the zymograms in man and was designated ADA ape. In all Pongidae the allele PGM ₁ ¹ was present, in addition in Gorilla g. gorilla a second allele was demonstrated, PGM ₁ ^Go . In Pan troglodytes a second allele, PGM ₁ ^Pan was recognized. In Pongo pygmaeus and Gorilla g. gorilla the PGM₂ patterns differed in their migration rates from PGM₂ 1 in man. In one individual of the species Pan troglodytes a PGM₂ zymogram was found resembling the heterozygous phenotype PGM₂ 3–1, PGM ₂ ¹ PGM ₂ ³, (type Palmer) in man. In all the other individuals of the species Pan troglodytes and in those of the species Pan paniscus the PGM₂ zymogram corresponded to the phenotype PGM₂ 1 in man.

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Zusammenfassung Bei 43 Vertretern der Familie Pongidae, Pongo pygmaeus (n=10), Gorilla g. gorilla (n=8), Pan troglodytes (n=22) und Pan paniscus (n=3), wurden die Erythrocytenenzyme saure Phosphatase, Adenylatkinase, Adenosindeaminase und Phosphoglucomutase mit der horizontalen Stärkegelelektrophorese analysiert. Bei allen Pongiden fanden wir eine saure Phosphatase, die dem Phänotyp B des Menschen entsprach, und eine Adenylatkinase, die dem Phänotyp AK 1 des Menschen glich. Alle Pongiden besaßen das gleiche, einem homozygoten Phänotyp entsprechende Adenosindeaminase-Zymogramm, das sich von den Zymogrammen des Menschen unterschied; wir bezeichnen diesen Phänotyp mit ADA ape. Bei allen Pongiden kommt das Allel PGM ₁ ¹ vor, bei Gorilla g. gorilla zusätzlich ein zweites Allel, PGM ₁ ^Go , und bei Pan troglodytes ein zweites Allel, PGM ₁ ^Pan . Die PGM₂-Zymogramme von Pongo pygmaeus und Gorilla g. gorilla unterschieden sich in ihrer elektrophoretischen Wandergeschwindigkeit vom Phänotyp PGM₂ 1 des Menschen. Bei einem Individuum der Species Pan troglodytes fanden wir ein heterozygotes PGM₂-Zymogramm, das an den heterozygoten Phänotyp PGM₂ 3–1, PGM ₂ ¹ PGM ₂ ³ (Typ Palmer) des Menschen erinnerte, bei allen übrigen Individuen der Species Pan troglodytes und bei denen der Species Pan paniscus ein homozygotes PGM₂-Zymogramm, das dem Phänotyp PGM₂ 1 des Menschen entsprach.

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Supported by the Deutsche Forschungsgemeinschaft.     

Interdependence of cell attachment and cell cycle signaling

Adult metazoans represent the culmination of an intricate developmental process involving the temporally and spatially orchestrated division, migration, differentiation, attachment, polarization and death of individual cells. An elaborate infrastructure connecting the cell cycle and cell attachment machinery is essential for such exquisite integration of developmental processes. Integrin-, cadherin-, Merlin- and planar cell polarity (PCP)-dependent signaling cascades quantitatively and qualitatively program cell division during development. Proteins in this signaling infrastructure may represent an important source of cancer vulnerability in metazoans, as their dysfunction can pleiotropically promote the oncogenic process.     

Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling

Resuscitation-promoting factor (Rpf) is a muralytic enzyme that increases the culturability of dormant bacteria. Recently, considerable progress has been made in understanding the structure, function and physiological role of Rpfs in different organisms, most notably the major human pathogen, Mycobacterium tuberculosis , which encodes multiple rpf -like genes. A key unresolved question, however, concerns the relationship between the predicted biochemical activity of Rpfs – cleavage of the β-1,4 glycosidic bond in the glycan backbone of peptidoglycan – and their effect on culturability. In M. tuberculosis , the interaction between RpfB and the d,l -endopeptidase, Rpf interacting protein A (RipA), enables these proteins to synergistically degrade peptidoglycan to facilitate growth. Furthermore, the combined action of Rpfs with RipA and other peptidoglycan hydrolases might produce muropeptides that could exert diverse biological effects through host and/or bacterial signaling, the latter involving serine/threonine protein kinases. Here, we explore these possibilities in the context of the structure and composition of mycobacterial peptidoglycan. Clearly, a deeper understanding of the role of Rpfs and associated peptidoglycan remodeling enzymes in bacterial growth and culturability is necessary to establish the significance of dormancy and resuscitation in diseases such as tuberculosis, which are associated with long-term persistence of viable bacterial populations recalcitrant to antibiotic and immune clearance.     

The relationship of red cell enzymes to red cell life-span

Red cells are replaced before they become senescent. It is probable that red cell destruction is controlled by a biologic clock, essentially independent of metabolism, rather than by metabolic failure. The appearance of neo-antigens on the external surface of the red cell could be this "clock."     

Giving space to cell signaling

Spatial separation of component enzymes in a pathway creates microdomains and gradients of signaling activities. Imaging techniques and computational modeling help us to understand the complex relationships between signaling and spatial information. In this issue, Neves et al. (2008) report how the propagation of spatial information is controlled by the shape of hippocampal neurons and feedback and feed-forward motifs of the beta-adrenergic receptor signaling pathway.     

Lysophospholipid receptors in cell signaling

There is increasing evidence that different phospholipids are involved in regulation of various cell processes and cell-cell interactions. Lysophospholipids (lysophosphatidic acid, lysophosphatidylcholine) and a number of lysosphingolipids play particular roles in these regulations. Their effects are mediated by specific G-protein-coupled receptors. G-Protein coupled signal transduction to the cell nucleus involving a chain of intracellular protein kinases induces the main effects in cells--growth, proliferation, survival, or apoptosis. This review summarizes recent data on various groups of lysophospholipid receptors and their cell signal transduction pathways.     

Antioxidant enzymes in renal cell carcinoma

The aim of the study was to estimate the significance of oxidative/nitrosative damage and expression of antioxidant enzymes in renal cell carcinomas (RCC). For this we investigated immunohistochemically six antioxidant enzymes (AOEs) including MnSOD, ECSOD, thioredoxin, thioredoxin reductase, and gammaglutamyl cysteine synthetase heavy and light chain in 138 RCCs. As an indicator of oxidative/nitrosative damage, sections were stained with an antibody to nitrotyrosine. The extent of apoptosis was evaluated by TUNEL method and proliferation by immunohistochemistry to Ki67. Variable expression of all AOEs could be seen in RCC with expression of MnSOD being strongest. Nitrotyrosine was significantly associated with high grade tumors. MnSOD was associated with tumors of a lower stage. Cases showing ECSOD reactivity had higher and cases expressing thioredoxin lower apoptotic index than other tumors. No association with patient prognosis was observed. According to the results renal cell carcinomas show oxidative/nitrosative damage which, according to nitrotyrosine staining, was higher in high grade tumors. Of AOEs, MnSOD was more abundantly expressed in low stage tumors suggesting that its antioxidant function could play a main role to prevent development of oxidative damage leading to more aggressive tumors.