Protein Targeting Protocols

It is by no means a revelation that proteins are not uniformly distributed throughout the cell. As a result, the idea that protein molecules, because of the specificity with which they can engage in interactions with other proteins, may be aimed—via these interactions—at a restricted target, is a fundamental one in contemporary molecular life sciences. The target may be variously c- ceived as a specific molecule, a group of molecules, a structure, or a more generic type of intracellular environment. Because the concept of protein targeting is intuitive rather than expl- itly defined, it has been variously used by different groups of researchers in cell biology, biochemistry, and molecular biology. For those working in the field of intracellular signaling, an influential introduction to the topic was the seminal article by Hubbard & Cohen (TIBS [1993] 18, 172–177), which was based on the work of Cohen’s laboratory on protein phosphatases. Sub- quently, the ideas that they discussed have been further developed and extended by many workers to other key intermediaries in intracellular sign- ing, including protein kinases and a great variety of modulator and adaptor proteins.

This volume contains a collection of innovative techniques for studying targeted protein degradation. Chapters guide readers through heterobifunctional proteolysis-targeting chimeras (PROTACs) approaches, E3 ligase, E3 ligase-induced ubiquitylation, proteomic approaches, novel degrader molecules, molecular glue, and stabilize binding interaction between a target and E3 ubiquitin ligase. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Targeted Protein Degradation: Methods and Protocols aims to ensure successful results in this emerging field of drug discovery.

Short Protocols in Protein Science provides condensed descriptions of more than 500 protocols compiled from Current Protocols in Protein Science. Drawing from both the original "core" manual as well as the quarterly update service, this compendium includes all step-by-step descriptions of the principal methods covered in Current Protocols in Protein Science.

With the recent completion of the sequencing of the human genome, it is widely anticipated that the number of potential new protein drugs and targets will escalate at an even greater rate than that observed in recent years. However, identification of a potential target is only part of the process in developing these new next generation protein-based “drugs” that are increasingly being used to treat human disease. Once a potential protein drug has been identified, the next rate-limiting step on the road to development is the production of sufficient authentic material for testing, charact- ization, clinical trials, and so on. If a protein drug does actually make it through this lengthy and costly process, methodology that allows the production of the protein on a scale large enough to meet demand must be implemented. Furthermore, large-scale production must not compromise the authenticity of the final product. It is also nec- sary to have robust methods for the purification, characterization, viral inactivation and continued testing of the authenticity of the final protein product and to be able to formulate it in a manner that retains both its biological activity and lends itself to easy administration. Therapeutic Proteins: Methods and Protocols covers all aspects of protein drug production downstream of the discovery stage. This volume contains contributions from leaders in the field of therapeutic protein expression, purification, characterization, f- mulation, and viral inactivation.

Protein Targeting, Transport, and Translocation presents an in-depth overview on the topic of protein synthesis, covering all areas of protein science, including protein targeting, secretion, folding, assembly, structure, localization, quality control, degradation, and antigen presentation. Chapters also include sections on the history of the field as well as summary panels for quick reference. Numerous color illustrations complement the presentation of material. This book is an essential reference for anyone in biochemistry and protein science, as well as an excellent textbook for advanced students in these and related fields. Basic principles and techniques Targeting adn sorting sequences Protein export in bacteria Membrane protein integration into ER and bacterial membranes Protein translocation across the ER Disulfide bond formation in prokaryotes and eukaryotes Quality control in the export pathway Import of proteins into organelles The secretory pathway Vesicular transport Spectacular color throughout

Protein Targeting reviews different aspects of protein targeting, including similarities and differences in the pathways involved. The early stages of protein export and secretion in prokaryotes and eukaryotes are examined, along with the targeting of mitochondrial and chloroplast proteins as well as some important differences in the biogenesis of the two organelles. Comprised of nine chapters, this book begins with an overview of terminology and basic principles of protein targeting, focusing on protein traffic in eukaryotic and bacterial cells. The reader is then introduced to the basic principles and techniques of protein targeting, including protein identification and posttranslational modification. Subsequent chapters deal with the early stages in the secretory pathway; later stages in the prokaryotic and eukaryotic secretory pathways; targeting of mitochondrial, chloroplast, and peroxisomal proteins; and targeting of nuclear proteins. The book also discusses endocytosis before concluding with a description of applications of protein targeting. This monograph will be of value to scientists and biotechnologists, as well as to students of cell biology, biochemistry, microbiology, and molecular biology.

Protein Phosphatase Protocols presents a broad range of protocols for the study of protein phosphatases, all written by experts and innovators from phosphatase laboratories around the world. This volume is a compendium of resources for the study of protein phosphatases and their potential as drug targets. Experimental methodologies are taken from proteomics, bioinformatics, genomics, biochemistry, RNAi, and genetics.

This volume reviews the increasing importance of glycosylation to the field of virology, as well as virus replication. The chapters provide an overview of glycosylation in relation to virus infection, and the generic techniques that are used to analyze and characterize glycoproteins. The information presented here provides insight as to how the techniques of glycobiology can be applied in virology and answer most questions that are of interest to the reader.

The molecular characterization of RNA and its interactions with proteins is an important and exciting area of current research. Organisms utilize a variety of RNA–protein interactions to regulate the expression of their genes. This is particularly true for eukaryotes, since newly synthesized messenger RNA must be extensively modified and transported to the cytoplasm before it can be used for protein synthesis. The realization that posttranscriptional processes are critical components of gene regulation has sparked an explosion of interest in both stable ribonucleoprotein (RNP) complexes and transient RNA–protein interactions. RNA is conformationally flexible and can adopt complex structures that provide diverse surfaces for interactions with proteins. The fact that short RNA molecules (aptamers; see Chapter 16) can be selected to bind many different types of molecules is evidence of the structural variability of RNA. RNA molecules are rarely entirely single- or double-stranded, but usually contain multiple short duplexes interrupted by single-stranded loops and bulges; in some RNAs, such as tRNAs, the short duplexes stack on each other. Further variability is generated by the presence of non-Watson-Crick base pairs, modified nucleotides, and more complex structures, such as pseudoknots and triple-strand interactions.