Alfred L. Goldberg

Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules

Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblasts, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules.

The ubiquitinproteasome pathway is required for processing the NF-κB1 precursor protein and the activation of NF-κB

Summary We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-κB. The p105 precursor of the p50 subunit of NF-κB is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-κB and the rapid degradation of IκBα induced by tumor necrosis factor α. Thus, the ubiquitinproteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.

Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy

Skeletal muscle atrophy is a debilitating response to fasting, disuse, cancer, and other systemic diseases. In atrophying muscles, the ubiquitin ligase, atrogin-1 (MAFbx), is dramatically induced, and this response is necessary for rapid atrophy. Here, we show that in cultured myotubes undergoing atrophy, the activity of the PI3K/AKT pathway decreases, leading to activation of Foxo transcription factors and atrogin-1 induction. IGF-1 treatment or AKT overexpression inhibits Foxo and atrogin-1 expression. Moreover, constitutively active Foxo3 acts on the atrogin-1 promoter to cause atrogin-1 transcription and dramatic atrophy of myotubes and muscle fibers. When Foxo activation is blocked by a dominant-negative construct in myotubes or by RNAi in mouse muscles in vivo, atrogin-1 induction during starvation and atrophy of myotubes induced by glucocorticoids are prevented. Thus, forkhead factor(s) play a critical role in the development of muscle atrophy, and inhibition of Foxo factors is an attractive approach to combat muscle wasting.

Protein degradation and protection against misfolded or damaged proteins

The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy.

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

Proteasome inhibitors: valuable new tools for cell biologists.

Proteasomes are major sites for protein degradation in eukaryotic cells. The recent identification of selective proteasome inhibitors has allowed a definition of the roles of the ubiquitin-proteasome pathway in various cellular processes, such as antigen presentation and the degradation of regulatory or membrane proteins. This review describes the actions of these inhibitors, how they can be used to investigate cellular responses, the functions of the proteasome demonstrated by such studies and their potential applications in the future.

Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression

Skeletal muscle atrophy is a debilitating response to starvation and many systemic diseases including diabetes, cancer, and renal failure. We had proposed that a common set of transcriptional adaptations underlie the loss of muscle mass in these different states. To test this hypothesis, we used cDNA microarrays to compare the changes in content of specific mRNAs in muscles atrophying from different causes. We compared muscles from fasted mice, from rats with cancer cachexia, streptozotocin‐induced diabetes mellitus, uremia induced by subtotal nephrectomy, and from pair‐fed control rats. Although the content of >90% of mRNAs did not change, including those for the myofibrillar apparatus, we found a common set of genes (termed atrogins) that were induced or suppressed in muscles in these four catabolic states. Among the strongly induced genes were many involved in protein degradation, including polyubiquitins, Ub fusion proteins, the Ub ligases atrogin‐1/MAFbx and MuRF‐1, multiple but not all subunits of the 20S proteasome and its 19S regulator, and cathepsin L. Many genes required for ATP production and late steps in glycolysis were down‐regulated, as were many transcripts for extracellular matrix proteins. Some genes not previously implicated in muscle atrophy were dramatically up‐regulated (lipin, metallothionein, AMP deaminase, RNA helicase‐related protein, TG interacting factor) and several growth‐related mRNAs were down‐regulated (P311, JUN, IGF‐1‐BP5). Thus, different types of muscle atrophy share a common transcriptional program that is activated in many systemic diseases.—Lecker, S. H., Jagoe, R. T., Gilbert, A., Gomes, M., Baracos, V., Bailey, J., Price, S. R., Mitch, W. E., Goldberg, A. L. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression.—Stewart H. Lecker, R. Thomas Jagoe, Alexander Gilbert, Marcelo Gomes, Vickie Baracos, James Bailey, S. Russ Price, William E. Mitch, Alfred L. Goldberg Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J. 18, 39–51 (2004)

Proteasome inhibitors: from research tools to drug candidates

The 26S proteasome is a 2.4 MDa multifunctional ATP-dependent proteolytic complex, which degrades the majority of cellular polypeptides by an unusual enzyme mechanism. Several groups of proteasome inhibitors have been developed and are now widely used as research tools to study the role of the ubiquitin-proteasome pathway in various cellular processes, and two inhibitors are now in clinical trials for treatment of multiple cancers and stroke.

Cellular Defenses against Unfolded Proteins: A Cell Biologist Thinks about Neurodegenerative Diseases

1993; Massa et al., 1996). Intracellular inclusions of de-natured proteins are also characteristic features of many neurological diseases, including Amyotrophic Lateral Sclerosis, Alzheimers disease, Parkinsons disease, and several hereditary diseases caused by expansions of polyglutamine tracts (e.g., Huntingtons Disease or the spinocerebellar ataxias) (Table 2). In all these neuro-Harvard Medical School 240 Longwood Avenue degenerative diseases, the pathology and the eventual death of specific neuronal populations occur due to the Boston, Massachusetts 02115 accumulation of distinct abnormal polypeptides. Many observations suggest that these various types of inclusions arise through common mechanisms and elicit sim-Introduction ilar host responses. For example, all these inclusions A continuous threat to cell function and viability is the contain components of the ubiquitin-proteasome degra-accumulation in cells of proteins with highly abnormal dative pathway and also molecular chaperones, which conformations. Protein structures, once formed, are not represent the two main systems that protect eukaryotic very stable chemical entities, and at 37ЊC, in the highly cells against the buildup of unfolded polypeptides. or certain antibiotics may seem to represent quite dif-occurring continuously. The rate of such damage to cell ferent challenges for a cell, but, in fact, they share the proteins can increase markedly upon exposure of cells capacity to cause severe damage to proteins. It is not sur-to harsh environmental conditions, such as increased prising, therefore, that common mechanisms emerged temperatures, oxygen free radicals, or heavy metals (es-early in evolution that enable cells to better withstand pecially mercury). Polypeptides with highly abnormal these diverse physical and chemical insults. Virtually all conformations may also result from mutations that pre-cells respond to these potentially toxic conditions by vent normal folding or prevent the association of a poly-induction of a set of highly conserved genes that encode peptide with other subunits or stabilizing cofactors. Be-heat shock proteins (Hsps). This set of proteins functions cause an accumulation of unfolded proteins can have as the major cellular defense against the accumulation very deleterious effects on cell function, all cells expend of damaged or mutant proteins. Among the Hsps in a significant fraction of their basal energy production to eukaryotic cells are many molecular chaperones, which ensure that proteins are accurately expressed, correctly function to retard protein denaturation and aggregation, folded, and targeted to the correct compartment. If na-several antioxidant enzymes, which reduce oxidative tive conformations are lost through mutation or postsyn-damage to cell proteins, and components of the ubiqui-thetic damage, cells have …

Multiple proteolytic systems, including the proteasome, contribute to CFTR processing

The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER.