Current Signal Transduction Therapy - Volume 5, Issue 1, 2010

The journal Current Signal Transduction Therapy will start its 5th year in 2010 and in the past four years it has covered a very broad spectrum of the signaling- related molecular pathomechanisms and the existing and potential therapeutic possibilities. The journal has been very well received by the scientific community as it has generated an impact in three years and it is acknowledged and cited in many scientific publications. The current aim of the journal is to become a leading journal and a unique forum for describing and analyzing the causes and the perspectives of signaling related diseases and therapies with special emphasis on recently emerged concepts as “multiple targets” and “novel targeted therapies”, “network signaling”, “molecular diagnostics”, “systems biology”, “personalized therapy” and “integrated drug and pharmacodiagnostic biomarker development”. If we want to understand signaling disorders for therapeutic intervention we have to get a deeper insight into these fields. So in the forthcoming years CSTT intends to cover various aspects of these signaling related areas and concepts. In the very complex area of network signaling and multiple targets we have to face the problem that the proteins encoded in the human genome are coordinated and regulated in a very complex way via protein-protein interactions and further posttranslational modifications including phosphorylation, glycosylation, sulphation, acetylation, ADP ribozylation and ubiquitinylation. These processes tremendously increase the heterogeneity of the proteome, which needs to be thoroughly analyzed to distinguish between causes and causatives from the pathological point of view and to identify the points of interference. The Editors of CSTT have decided to dedicate certain issues of the journal for such hot topics in signaling and signal transduction therapy. Glycosylation of nuclear and cytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides has been identified recently as a very important, abundant posttranslational modification. So the current issue of CSTT is dedicated to glycosylation particularly O-GlcNAc modification of proteins which seems to be a very important signaling mechanism of proteins and actually it seems to be a complementary signal for phosphorylation. Prof. Brigitte Schmitz from the University of Bonn, one of the outstanding experts of the field has accepted our invitation to act as guest editor for this “hot-topic” issue and she has brought along a highly respected group of scientists to provide a really comprehensive and detailed overview of the role of O-GlcNAc modification of the proteins in signaling and signaling disorders. We are confident that this hot-topic issue will be very interesting and informative for the scientific community interested in novel aspects of signaling and signal transduction therapy.

It appears to be now fairly well accepted in the field of signal transduction that the monosaccharide N-acetylglucosamine (O-GlcNAc) added posttranslationally through a β-O-glycosidic linkage to serine and threonine residues in cytosolic and nuclear proteins has important regulatory roles. Since its first description in 1984 until today this highly dynamic sugar has been shown to be attached to about thousand proteins. The steady increase in the number of published papers in recent years which deal with O-GlcNAc underlines the importance of O-GlcNAc in most, if not all processes believed in the past to be regulated merely by serine/threonine phosphorylation. But nevertheless, many investigators - who are well aware of phosphorylation as a regulatory modification in processes as diverse as the cell cycle or glycogen metabolism - are still surprised to learn that a sugar residue might act in a similar way, although often in a reciprocal fashion to phosporylation. In a review article on the occasion of the 20th anniversary of the discovery of O-GlcNAc Gerald Hart commented on how long it took for protein phosphorylation to become accepted as an important regulatory modification of proteins from its discovery in 1933. If one considers the mere quantitative output, based on the number of publications concerned with OGlcNAc, one notices that considerably more papers have been published in the last five years than in the twenty years from 1984 - 2004. So there are grounds to hope that general acceptance of O-GlcNAc as a regulatory modification will not take as long as for phosphorylation. Difficulties in detecting O-GlcNAc modification on proteins and in particular in identifying sites of modification have contributed significantly to the initial slow progress in understanding the functions of O-GlcNAc. Recent developments of innovative chemo-enzymatic, as well as mass spectrometric and other techniques have, however, changed this situation greatly. These advances have been extremely important in gaining deeper insights into the function of O-GlcNAc in physiological processes and pathological conditions, such as diabetes type 2, cardiovascular and neurodegenerative diseases, or cancer. I would like to thank the Editors in Chief of CSTT - as will certainly all other O-GlcNAc-“Fans” - for giving me the opportunity of raising the prominence of O-GlcNAc through this Journal, and of giving interested researchers the opportunity of obtaining a concise overview of this exciting and fascinating aspect of signal transduction. It is obvious that O-GlcNAc, as well as the enzymes which catalyse its rapid turnover, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), are excellent targets for signal tranduction therapy. It is to be hoped that this special issue of CSTT will contribute to progress in this important research area, too. In each chapter of this issue authors present specific contributions to the elucidation of the role of O-GlcNAc, in addition to giving an overview of some more general O-GlcNAc-related aspects of processes in which O-GlcNAc is known to be implicated. In Chapter 1 the authors describe the role of O-GlcNAc as a nutrient sensor regulating energy metabolism, not only under diabetic but also under normo- and hypoglycemic conditions. After the discovery of the involvement of the hexosamine pathway in insulin resistance it took another seven years before it was demonstrated that the O-GlcNAc modification of proteins - one of several end-products of this pathway - is causally involved in aberrant insulin signalling. The authors of Chapter 1 and others confirmed much of the early work on glucosamine that its effects were due to increased/decreased OGlcNAcylation of proteins, by over-expressing, for example, either OGT or OGA in vitro or in vivo. The molecular mechanisms of how specific metabolic pathways are regulated by O-GlcNAcylation in addition to those of the better known phosphorylation reactions are described in this chapter as well supporting the view that acute hormone-mediated signalling can be attenuated resulting in cells protecting themselves from excessive nutrient flux or nutrient deprivation. The rapid and dynamic attachment or removal of O-GlcNAc (“O-GlcNAc cycling”) is not the only possible way of how OGlcNAc can regulate cellular functions. O-GlcNAc modification of RNA polymerase II and its transcription factors were discovered early on. In Chapter 2, an overview is given of the wealth of information regarding the regulation of different stages of transcription by O-GlcNAc from chromatin remodelling to e.g. proteasomal degradation of transcription factors supporting that O-GlcNAc has important roles in controlling gene expression in response to glucose levels. A description of recently developed techniques for O-GlcNAc site-mapping is also included in this chapter and, in addition, of screening methods such as two hybrid assays capable of providing much more data on O-GlcNAcylation more rapidly than conventional analyses. These two aspects are also central to Chapter 3. The authors of the other reviews will forgive me for mentioning the last author of this chapter here. Gerald Hart is the pioneer in this exciting research area and he and his coworkers have probably worked in most of the areas in which O-GlcNAcylation plays a role, including cloning of the enzymes OGT and OGA as well as the development of methods for identification and site mapping. His comprehensive review makes clear that O-GlcNAc's roles are essential for most vital cellular functions in healthy situations as well as being involved in many diseases. The power of comparative O-GlcNAc proteome studies, e.g., between normal and diabetic patients, has been demonstrated recently by his group and it is likely that data of such a study could provide the basis for a diagnostic tool for this disease. In this chapter data are shown from a comparative O-GlcNAc proteome study of resting and activated T-cells using SILAC in combination with immunoaffinity enrichment and mass spectrometry. In addition to known O-GlcNAc functions these data reveal new functions as well as having important regulatory implications for T-cell activation. Similar O-GlcNAc proteome approaches are now being carried out more and more, thus catching up with site-mapping and functional analysis of phosphorylation. In Chapter 4 the role of O-GlcNAc in immune regulation is presented with particular emphasis on the role of O-GlcNAcmodified transcription factors during reprogramming of activated T- and B-cells leading to their maturation. Although the link between T- or B-cell receptor activation and enhanced O-GlcNAc modification on transcription factors is not yet clear, nuclear translocation appears to be at least one step requiring O-GlcNAcylation. Altogether, it appears that decreasing O-GlcNAc levels could reduce responses by the adaptive immune system, which would be beneficial in autoimmune diseases but might facilitate virus replication, e.g., in HIV. On the other hand, in the innate immune response increased O-GlcNAc correlates with enhanced neutrophil motility and protection against inflammation. This view parallels evidence presented in Chapter 5 on the cardiovascular system. Increased O-GlcNAcylation mediates at least a short term cytoprotective effect. This is in accord with earlier observations that the O-GlcNAc level on proteins is increased in response to several stress stimuli. Combining these observations made in vitro and in more clinically relevant in vivo models of myocardial infarction with those pathophysiological effects of enhanced O-GlcNAc in diabetes described in Chapter 1, leads to the hypothesis that the initial response to acute stress produces activation of protective pathways including O-GlcNAc, whereas long lasting activation of the same pathway(s) may result in chronic adverse and pathophysiological effects. A highly promising approach to understanding O-GlcNAc's multiple functions in health and disease and its exploitation for therapeutic application is described in Chapter 6. The nematode Caenorhabditis elegans is an excellent model system allowing rapid large scale genomic and proteomic analyses for studying interactions and signalling pathways for O-GlcNAc modified proteins in wild-type and deletion mutants. Besides the study of nutrient sensing and known signalling pathways in which OGlcNAc participates, the discovery of as yet unknown functions of O-GlcNAc and O-GlcNAc's relationships to other signalling networks through systematic genetic interaction analysis provides a tool for investigating novel connections with other genetic networks. The combinatorial use of these techniques, together with the use of C. elegans high throughput screens, has the potential for revealing valuable data on O-GlcNAc essential for increasing knowledge and for therapeutic development. Finally, to understand precisely the mechanisms by which the enzymes OGT and OGA can recognize and catalyze the cycling of O-GlcNAc on probably thousands of proteins without identifiable consensus sequences, is yet another outstandingly important issue. In Chapter 7 mechanistic, structural and kinetic studies are described which address this and related questions. For several reasons much more is known of the structure and catalytic mechanism of OGA. In recent years highly selective inhibitors of OGA have been designed which are also applicable in vivo, as demonstrated in a study proving that a specific inhibitor can pass the blood brain barrier and is capable of reducing hyperphoshorylation of the tau protein, one of the pathological hallmarks of Alzheimer's disease. Structural studies and investigation of the mechanism of enzymatic action for OGT, as well as the search for selective inhibitors for this enzyme are currently subjects of intense research. Results are awaited with high expectations for progress in gaining deeper insights and in developing therapeutic applications. To conclude I should like to thank all those colleagues who have contributed to this Special Issue for providing a cuttingedge, topical and comprehensive review of the importance and biological significance of protein O-GlcNAcylation.

Modification of cytosolic, nuclear, and mitochondrial proteins on serine and threonine residues by Nacetylglucosamine (O-GlcNAc) provides a mechanism by which glucose flux rates in the cell can feed back to regulate the function and synthesis of proteins involved in metabolism and growth. The hexosamine biosynthesis pathway (HBP) provides the substrate for this modification, UDP-GlcNAc. Production of UDP-GlcNAc and subsequent protein modifications are generally reflective of glucose flux rates at higher than normal glucose levels, hence the pathway serves a nutrient sensing function. At lower than normal glucose levels, at least some cells upregulate the enzyme responsible for the modification, O-linked GlcNAc transferase (OGT), such that levels of O-GlcNAc on protein also increases at lower than normal glucose concentrations. We summarize herein the evidence that the HBP/O-GlcNAc pathways have an important role in metabolic regulation, both in normal physiology and in pathologic states of high glucose levels. Examples are provided of how the modification alters function of important metabolic enzymes and transcription factors involved in metabolic regulation in muscle, fat, liver, and pancreatic β-cells. The evidence supporting the importance of the pathway is such that any study of metabolic regulation-by mechanisms that are transcriptional, translational, or posttranslational, or through modulation of protein sequestration or degradation-should investigate the possible role of the pathway in that process.

O-linked a-N-acetylglucosamine (O-GlcNAc) modification of proteins has been shown to be involved in many different cellular processes, such as cell cycle control, nutrient sensing, signal transduction, stress response and transcriptional regulation. Cells have developed complex regulatory systems in order to regulate gene expression appropriately in response to environmental and intracellular cues. Control of eukaryotic gene transcription often involves post-translational modification of a multitude of proteins including transcription factors, basal transcription machinery, and chromatin remodeling complexes to modulate their functions in a variety of manners. In this review we describe the emerging functional roles for and techniques to detect and modulate the O-GlcNAc modification and illustrate that the O-GlcNAc modification is intricately involved in at least seven different general mechanisms for the control of gene transcription.

GlcNAcylation is a dynamic cytoplasmic and nuclear post-translational sugar modification of serine/threonine residues. The addition and removal of O-GlcNAc are regulated by O-GlcNAc Transferase and O-GlcNAcase, respectively. Over ∼1000 proteins have been identified to be GlcNAcylated with over 240 mapped sites. O-GlcNAc is involved in critical cellular functions, such as cell-cycle regulation, apoptosis, stress responses, signaling, transcription, and translation. O-GlcNAc also plays pivotal roles in diseases, such as diabetes, neurodegenerative disease and cancer, and immunological regulation, such as T-cell activation. Through comparative proteomic analysis of resting and activated T-cells, we identified potentially GlcNAcylated proteins involved in post-signaling events of T-cell activation. O-GlcNAc on 58 proteins involved in processes, such as DNA replication, cytoskeletal rearrangement, chromatin remodeling, and RNA processing, were altered by T-cell activation. GlcNAcylation and phosphorylation are similar in abundance and cellular/ biological function, and their regulation is deeply intertwined. The two modifications regulate each other at the sitelevel by reciprocally influencing site-occupancy, and at the enzymatic-activity level by each modification modulating the catalytic activity of the enzymes involved in the other modification. This paper will focus on recent developments in the interplay between O-GlcNAc and phosphorylation, and O-GlcNAc's roles in human disease and immunology.

The posttranslational modification of proteins by a single O-linked N-acetylglucosamine (O-GlcNAc) is an intracellular biochemical reaction which is ubiquitous in eukaryotic cells. Two enzymes regulate the process: O-linked Nacetylglucosaminyltransferase (OGT), which attaches O-GlcNAc to serine/threonine residues of proteins, and a a-Nacetylglucosaminidase (O-GlcNAcase), that removes the O-GlcNAc group. The serine or threonine targeted by OGlcNAc can occur at sites modified by other enzymes such as protein kinases. There have been many indications that OGlcNAc modifications are actively involved in the regulation of the immune system and recent results have begun to shed light on possible mechanisms. This review summarizes recent advances in the field of O-GlcNAc modification, with a special attention to its role in the activation of lymphocytes.

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms.

Caenorhabditis elegans is perhaps the best-understood metazoan in terms of cell fate, neural connectivity, nutrient sensing, and longevity. The study of this genetically amenable model has greatly accelerated progress in understanding human aging-associated diseases, such as diabetes and neurodegeneration. The nutrient-responsive cycling of OGlcNAc on key intracellular targets may play a key, yet unappreciated, role in human disease. Unlike their mammalian counterparts, loss-of-function mutants of ogt-1 (O-GlcNAc Transferase) and oga-1 (O-GlcNAcase) are viable in C. elegans, allowing the impact of the loss of O-GlcNAc cycling to be monitored in a living organism. C. elegans forward and reverse genetics, coupled with proteomics and chemical genomics, reveal networks of interactions and signaling pathways in which O-GlcNAc cycling may participate. The results point to a key regulatory role for O-GlcNAc cycling in cellular functions as diverse as nutrient uptake and salvage, cellular signaling, and transcription. The impact of altered O-GlcNAc cycling on the organism includes many of the hallmarks of aging-associated diseases: altered metabolism, lifespan, stress resistance, and immunity.

The post-translational modification of nucleocytoplasmic proteins with O-linked 2-acetamido-2-deoxy-Dglucopyranose (O-GlcNAc) is a topic of considerable interest and attracts a great deal of research effort. O-GlcNAcylation is a dynamic process which can occur multiple times over the lifetime of a protein, sometimes in a reciprocal relationship with phosphorylation. Several hundred proteins, which are involved in a diverse range of cellular processes, have been identified as being modified with the monosaccharide. The control of the O-GlcNAc modification state on different protein targets appears to be important in the aetiology of a number of diseases, including type II diabetes, neurodegenerative diseases and cancer. Two enzymes are responsible for the addition and removal of the O-GlcNAc modification: uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyltransferase (OGT) and O-GlcNAcase (OGA), respectively. Over the past decade the volume of information known about these two enzymes has increased significantly. In particular, mechanistic studies of OGA, in conjunction with structural studies of bacterial homologues of OGA have stimulated the design of inhibitors and offered a rationale for the binding of certain potent and selective inhibitors. Mechanistic information about OGT lags a little way behind OGA, but the recent deduction of the structure of an OGT bacterial homologue should now drive these studies forward.