Ubiquitination and Protein Stability - Part B, Volume 619, the latest release in the Methods in Enzymology series, highlights new advances in the field, with this updated volume presenting interesting chapters written by an international board of authors. Topics of note include chapters on Assays of SUMO protease function in mammalian cells, In vitro analysis of proteasome-associated USP14 activity for substrate degradation and deubiquitylation, Methods to study proteasome regulatory particle assembly, Native mass spectrometry approaches to study the proteasome, Single-molecule methods to study the ubiquitin-proteasome system, Assays for the function of ubiquitin in the mammalian endocytic pathway, and much more.

The last several years have been a landmark period in the ubiquitin field. The breadth of ubiquitin's roles in cell biology was first sketched, and the importance of ubiquitin-dependent proteolysis as a regulatory mechanism gained general acceptance. The many strands of work that led to this new perception are re counted in this book. A consequence of this progress is that the field has grown dramatically since the first book on ubiquitin was published almost a decade ago [M. Rechsteiner (ed. ), Ubiquitin, Plenum Press, 1988]. In this span, students of the cell cycle, transcription, signal transduction, protein sorting, neuropathology, cancer, virology, and immunology have attempted to chart the role of ubi quit in in their particular experimental systems, and this integration of the field into cell biology as a whole continues at a remarkable pace. We hope that for active researchers in the field as well as for newcomers and those on the fence, this book will prove helpful for its breadth, historical perspective, and practical tips. Structural data are now available on many of the components of the ubiquitin pathway. The structures have provided basic insights into the unusual biochemical mechanisms of ubiquitination and proteasome-mediated proteolysis. Because high-speed computer graphics can convey structures more effectively than print media, we have supplemented the figures of the book with a Worldwide Web site that can display the structures in a flexible, viewer-controlled format.

This volume gives an overview of pro tea some-mediated protein degradation and the regulatory role of the ubiquitin system in cellular proteolysis. The first chapter describes the molecular evolution of the proteasome and its associated activators, i. e. , the 20S core, the base and the lid of the 19S cap, and the 11 S regulator. The ensuing chapter gives an overview of the structure and assembly of the 20S proteasome and the regulation of the archaeal proteasome by PAN. The third contribution summarizes our knowledge on the eukaryotic 26S proteasome and its regulation by the 19S regu lator, followed by a chapter devoted to the llS regulator, which elucidates the structural basis for the 11 S-mediated activation of the 20S proteasome. The fifth chapter reviews in detail the role of the proteasome in the immune response. The subsequent chapter of the natural substrates of the gives a comprehensive description proteasome and their recognition by the enzymes of the ubiqui tination machinery. The penultimate chapter rounds up the in formation on intracellular distribution of proteasomes in yeast and mammalian cells, while the last contribution highlights proteasome inhibitors, tools which proved to be very valuable for dissecting the cellular roles of the proteasome and which might turn out to be of pharmacological importance.

A panel of leading academic and pharmaceutical investigators takes stock of the remarkable work that has been accomplished to date with proteasome inhibitors in cancer, and examines emerging therapeutic possibilities. The topics range from a discussion of the chemistry and cell biology of the proteasome and the rationale for proteasome inhibitors in cancer to a review of current clinical trials underway. The discussion of rationales for testing proteasome inhibitors in cancer models covers the role of the proteasome in NF-kB activation, the combining of conventional chemotherapy and radiation with proteasome inhibition, notably PS-341, new proteasome methods of inhibiting viral maturation, and the role of protesome inhibition in the treatment of AIDS. The authors also document the development of bortezomib (VelcadeTM) in Phase I clinical trials and in a multicentered Phase II clinical trials in patients with relapsed and refractory myeloma.

This volume brings together a set of reviews that provide a summary of our current knowledge of the proteolytic machinery and of the pathways of protein breakdown of prokaryotic and eukaryotic cells. Intracellular protein degradation is much more than just a mechanism for the removal of incorrectly folded or damaged proteins. Since many short-lived proteins have important regulatory functions, proteolysis makes a significant contribution to many cellular processes including cell cycle regulation and transciptional control. In addition, limited proteolytic cleavage can provide a rapid and efficient mechanism of enzyme activation or inactivation in eukaryotic cells. In the first chapter, Maurizi provides an introduction to intracellular protein degradation, describes the structure and functions of bacterial ATP-dependent proteases, and explores the relationship between chaperone functions and protein degradation. Many of the principles also apply to eukaryotic cells, although the proteases involved are often not the same. Interestingly, homologues of one of the bacterial proteases, Ion protease, have been found in mitochondria in yeast and mammals, and homologues of proteasomes, which are found in all eukaryotic cells (see below), have been discovered in some eubacteria. Studies of proteolysis in yeast have contributed greatly to the elucidation of both lysosomal (vacuolar) and nonlysosomal proteolytic pathways in eukaryotic cells. Thumm and Wolf (chapter 2) describe studies that have elucidated the functions of proteasomes in nonlysosomal proteolysis and the contributions of lysosomal proteases to intracellular protein breakdown. Proteins can be selected for degradation by a variety of differen mechanisms. The ubiquitin system is one complex and highly regulated mechanism by which eukaryotic proteins are targetted for degradation by proteosomes. In chapter 3, Wilkinson reviews the components and functions of the ubiquitin system and considers some of the known substrates for this pathway which include cell cycle and transcriptional regulators. The structure and functions of proteosomes and their regulatory components are described in the two subsequent chapters by Tanaka and Tanahashi and by Dubiel and Rechsteiner. Proteasomes were the first known example of threonine proteases. They are multisubunit complexes that, in addition to being responsible for the turnover of most short-lived nuclear and cytoplasmic protein, are also involved in antigen processing for presentation by the MHC class I pathway. Recent studies reviewed by McCracken and colleagues (chapter 6) lead to the exciting conclusion that some ER-associated proteins are degraded by cytosolic proteasomes. Lysosomes are responsible for the degradation of long-lived proteins and for the enhanced protein degradation observed under starvation conditions. In chapter 7 Knecht and colleagues review the lysosomal proteases and describe studies of the roles of lysosomes and the mechanisms for protein uptake into lysosomes. Methods of measuring the relative contribution of different proteolytic systems (e.g., ubiquitin-proteasome pathway, calcium-dependent proteases, lysosomes) to muscle protein degradation, and the conclusions from such studies, are reviewed by Attai and Taillinder in the following chapter. Finally, proteases play an important role in signaling apoptosis by catalyzing the limited cleavage of enzymes. Mason and Beyette review the role of the major players, caspases, which are both activated by and catalyze limite proteolysis, and also consider the involvement of other protoelytic enzymes in this pathway leading cell death.

The plant hormone auxin plays a fundamental role in the growth and development of plants. Researchers from across the globe are currently attempting to unravel the molecular mechanisms by which auxin controls such diverse processes as cell division, cell elongation, and differentiation. Research questions on auxin action are being addressed using state-of-the-art techniques that are available to cell biologists, geneticists, molecular biologists, biochemists, and physiologists. This text highlights many of the major topics that were covered in a recent workshop that was specifically focused on research into the mechanisms of auxin action. The articles in this text give a current update of the research findings on auxin biosynthesis, metabolism and transport; evolutionary patterns; auxin perception, signal transduction and physiology; auxin-regulated gene expression and protein degradation pathway in auxin responses; and cross-talk between auxin and other plant signalling pathways.