Protein misfolding and disease: Principles and protocols. Methods in molecular biology, Vol. 232, edited by P. Bross and N. Gregersen.

The past decade has seen a very large increase in the description of diseases as protein misfolding or conformational disorders. Folding of the protein chain to give the functional structure, and maintenance of the functional conformation are complex and critical processes. Our understanding of the impact of alternative conformers on the cell, and how alternative conformations may compromise cellular activity or induce toxicity, is steadily growing. Biophysical methods have been relatively successful at probing the structural consequences of some disease-associated mutations; for example, three-dimensional structures of variant proteins show the impact of particular mutations, analysis of altered folding and unfolding kinetics may illustrate the affect of a mutation on protein stability in vitro, and fiber diffraction and electron microscopy studies have illuminated the structure of amyloid fibrils. This book focuses on the study of the many cellular consequences of protein misfolding. It is a guide to the techniques that have been used most successfully to highlight differences between normal and disease-related processes. It offers a tour through past successful investigations and will challenge current researchers in the field to reassess the possibilities and to apply new strategies.

The three main categories of nonnative conformations—namely, stable misfolded forms, unstable misfolded forms, and aggregation-prone forms—can have three different consequences: functional deficiency, dominant-negative effects, or toxic cellular effects. The first studies of protein misfolding pathologies tended to focus most on intra- or extracellular aggregation of proteins in diseases that exhibit a gain-of-function pathology. There is increasing recognition that early species may be toxic in these processes, and there is now a shift toward investigating the cellular response to aggregation, in addition to studying the impact of the aggregation itself. Another large group of diseases involves the rapid degradation of mutant protein, resulting in a loss-of-function pathology. Increasingly, studies of different conformational diseases are highlighting the fact that the cellular responses observed with various pathologies have common features. Examination of these defective folding disorders has also highlighted the normal cellular mechanisms for dealing with protein quality control. One particular area that is increasingly well understood, and that is illuminated by studies of diseases associated with misfolding, is that of molecular chaperones and the proteolytic degradation systems such as the proteasome.

The editors present general concepts in the study of conformational diseases as an introduction to the field and have assembled descriptions of some of the best understood systems to illustrate the range of possible issues. These include cystic fibrosis with preventative degradation of variant proteins, and aggregation-associated problems such as Parkinson’s disease (PD) and α1-antitrypsin–associated retention of mutant protein in the endoplasmic reticulum. The General Methods section covers expression of recombinant protein, a discussion of the advantages and disadvantages of the systems available, and the use of site-directed mutagenesis and pulse-chase labeling techniques to probe the effect of disease-associated mutations on protein stability and turnover. These are techniques that have very broad application in many of the studies of conformational disease.

The description of what is known about cystic fibrosis transmembrane regulator (CFTR) misfolding and degradation in cystic fibrosis is thorough, based on extensive work by a number of groups. An important and interesting section is the discussion of the correction of the folding defect as a target for CFTR therapy. The central role played by the cellular quality control mechanisms is illustrated by the methods for studying endoplasmic reticulum (ER)-associated degradation (ERAD) and ubiquitination in CFTR literature.

The value of this volume lies in the detailed description of particular techniques that are specific to the studies of different disease states but which, taken together, add to the growing realization that common mechanisms are involved in conformational diseases. In the case of α₁antitrypsin deficiency (α₁AT), the studies of the fate of α₁AT and mutant α ₁AT in the ER and the elucidation of the cellular response to ER retention open up a new knowledge of the course of protein transport in the ER, both under normal and stress conditions. In PD, two different routes of cellular compromise have been identified: aggregation of α-synuclein and formation of neuronal inclusions, and also loss of activity of parkin, a member of the RING-finger containing E3 ubiquitin ligase family. Loss of parkin function is associated with selective degeneration of dopaminergic neurons. This work also highlights the importance of the unfolded protein stress (UPS) response, a key feature in the study of the cellular consequences of protein misfolding. The wide scope of the book includes aberrant protein folding in cancer. Here misfolding may be due to mutations that result in inactive tumor suppressors, or alternate conformations that are regulated differently or actually lead to dominant-negative inactivation of wild-type tumor suppressors or to constitutive activation of an oncogenic protein.

Studies of protein misfolding use the techniques applied to probing normal protein turnover and regulation and are often illuminated by comparison with the investigation of the mechanism of normal cellular responses. Investigation of the phenomenon of yeast prion proteins has shed light on the protein-only hypothesis in prion-associated diseases in animals. Yeast systems are well described in the book, both as expression systems and as systems in which to study misfolding. A number of strains are available that are defective for the activities of specific chaperones, making them very useful for probing the role of chaperones and proteasome degradation in certain pathologies.

The second half of the book covers particular studies of different diseases and the tools that have been applied to gain insight in different cases. For example, the use of substrates or chemical chaperones to rescue protein folding defects in human multidrug resistance P-glycoprotein (P-gp) and the use of two-hybrid systems to probe defective subunit interactions in the homotetrameric protein phenylalanine hydroxylase and the heteromeric mitochondrial protein propionyl-CoA carboxylase. Many of the protocols could be found elsewhere in general biochemical and cell biology reference texts, but the specific, context-related examples are what will challenge readers to apply these methods to their own particular area of the field. The general themes are protein expression, stability, processing, and degradation. The general tools include detection of protein aggregates, immunofluorescence and immunohistochemistry, cell culture, temperature dependence as an indicator of protein stability, pulse-chase methods, and antibody detection. In vitro synthesis of proteins is well covered, including a discussion of the study of protein synthesis and turnover in microsomes, mitochondria, or semipermeabilized cells.

Some contributors touch on the problem of the “knock-on” effect of having the cellular chaperone and proteasome machinery taken up with dealing with mutant protein, perhaps allowing other problems to slip though the net. One area that is not discussed is how the cell may deal with natively unfolded proteins. Many of these proteins have critical roles in signaling and cell cycle control, so problems with stability and turnover would have large consequences. However, most of what we currently understand about the cellular quality control mechanisms is directed at correct folding of globular proteins and the maintenance of that functional structure. Increasingly, we may learn more about how the cell regulates the stability and activity of natively unfolded proteins (NUPs). How can the usual assessments of quality control be applied to these proteins, when gain of stable folded structure cannot be used as a marker of quality?

This volume is part of the Methods in Molecular Biology series, and it is predominantly a practical handbook. Researchers who are working in the field of protein misfolding and disease will find in it detailed protocols for methods they already know they want to use and, hopefully, will be provoked to look at their problem with alternative approaches. Rather than giving a description of solving of the protein folding puzzles, the chapters in the book describe the tools used to unravel them.