Intracellular protein degradation: From a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting
Introduction
The concept of protein turnover is hardly 60 years old. Beforehand, body proteins were viewed as essentially stable constituents that were subject to only minor ‘wear and tear’: dietary proteins were believed to function primarily as energy-providing fuel, which were independent from the structural and functional proteins of the body. The problem was hard to approach experimentally, as research tools were not available. An important research tool that was lacking at that time were stable isotopes. While radioactive isotopes were developed earlier by George de Hevesy (de Hevsey G., Chemistry 1943. In: Nobel Lectures in Chemistry 1942–1962. World Scientific 1999. pp. 5–41), they were mostly unstable and could not be used to follow metabolic pathways). The concept that body structural proteins are static and the dietary proteins are used only as a fuel was challenged by Rudolf Scheonheimer in Columbia University in New York city. Schoenheimer escaped from Germany and joined the Department of Biochemistry in Columbia University founded by Hans T. Clarke [1], [2], [3]. There he met Harold Urey who was working in the Department of Chemistry and who discovered deuterium, the heavy isotope of hydrogen, a discovery that enabled him to prepare heavy water, D2O. David Rittenberg who had recently received his PhD in Urey's laboratory, joined Schoenheimer, and together they entertained the idea of “employing a stable isotope as a label in organic compounds, destined for experiments in intermediary metabolism, which should be biochemically indistinguishable from their natural analog” [1]. Urey later succeeded in enriching nitrogen with 15N, which provided Schoenheimer and Rittenberg with a “tag” for amino acids and as a result for the study of protein dynamics. They discovered that following administration of 15N-labled tyrosine to rat, only ∼50% was recovered in the urine, “while most of the remainder is deposited in tissue proteins. An equivalent of protein nitrogen is excreted’” [4]. They further discovered that from the half that was incorporated into body proteins “only a fraction was attached to the original carbon chain, namely to tyrosine, while the bulk was distributed over other nitrogenous groups of the proteins” [4], mostly as an αNH2 group in other amino acids. These experiments demonstrated unequivocally that the body structural proteins are in a dynamic state of synthesis and degradation, and that even individual amino acids are in a state of dynamic interconversion. Similar results were obtained using 15N-labled leucine [5]. This series of findings shattered the paradigm in the field at that time that: (1) ingested proteins are completely metabolized and the products are excreted, and (2) that body structural proteins are stable and static. Schoenheimer was invited to deliver the prestigious Edward K. Dunham lecture at Harvard University where he presented his revolutionary findings. After his untimely tragic death in 1941, his lecture notes were edited by Hans Clarke, David Rittenberg, and Sarah Ratner, and were published in a small book by Harvard University Press. The editors called the book, The Dynamic State of Body Constituents [6], adopting the title of Schoenheimer's presentation. In the book, the new hypothesis is clearly presented: “The simile of the combustion engine pictured the steady state flow of fuel into a fixed system, and the conversion of this fuel into waste products. The new results imply that not only the fuel, but the structural materials are in a steady state of flux. The classical picture must thus be replaced by one which takes account of the dynamic state of body structure.” However, the idea that proteins are turning over was not accepted easily and was challenged as late as the mid-1950s. For example, Hogness and colleagues studied the kinetics of β-galactosidase in E coli and summarized their findings [7]: “To sum up: there seems to be no conclusive evidence that the protein molecules within the cells of mammalian tissues are in a dynamic state. Moreover, our experiments have shown that the proteins of growing E coli are static. Therefore, it seems necessary to conclude that the synthesis and maintenance of proteins within growing cells is not necessarily or inherently associated with a ‘dynamic state’.” While the experimental study involved the bacterial β-galactosidase, the conclusions were broader, including also the authors' hypothesis on mammalian proteins. The use of the term ‘dynamic state’ was not incidental, as they challenged directly Schoenheimer's studies.
Now, after more than 6 decades of research in the field and with the discovery of the lysosome and later the complex ubiquitin-proteasome system with its numerous tributaries, it is clear that the area has been revolutionized. We now realize that intracellular proteins are turning over extensively, that this process is specific, and that the stability of many proteins is regulated individually and can vary under different conditions. From a scavenger, unregulated and non-specific end process, it has become clear that proteolysis of cellular proteins is a highly complex, temporally controlled and tightly regulated process that plays major roles in a broad array of basic pathways. Among these processes are cell cycle, development, differentiation, regulation of transcription, antigen presentation, signal transduction, receptor-mediated endocytosis, quality control, and modulation of diverse metabolic pathways. Subsequently, it has changed the paradigm that regulation of cellular processes occurs mostly at the transcriptional and translational levels, and has set regulated protein degradation in an equally important position. With the multitude of substrates targeted and processes involved, it is not surprising that aberrations in the pathway have been implicated in the pathogenesis of many diseases, among them certain malignancies, neurodegeneration, and disorders of the immune and inflammatory system. As a result, the system has become a platform for drug targeting, and mechanism-based drugs are currently developed, one of them is already on the market.
Section snippets
The lysosome and intracellular protein degradation
In the mid-1950s, Christian de Duve discovered the lysosome (see, for example, Refs. [8], [9] and Fig. 1). The lysosome was first recognized biochemically in rat liver as a vacuolar structure that contains various hydrolytic enzymes which function optimally at an acidic pH. It is surrounded by a membrane that endows the contained enzymes latency that is required to protect the cellular contents from their action (see below). The definition of the lysosome has been broadened over the years. This
The lysosome hypothesis is challenged
As mentioned above, the unraveled mechanism(s) of action of the lysosome could explain only partially, and at times not satisfactorily, several key emerging characteristics of intracellular protein degradation. Among them were the heterogeneous stability of individual proteins, the effect of nutrients and hormones on their degradation, and the dependence of intracellular proteolysis on metabolic energy. The differential effect of selective inhibitors on the degradation of different classes of
The ubiquitin-proteasome system
The cell-free proteolytic system from reticulocytes [28], [29] turned out to be an important and rich source for the purification and characterization of the enzymes that are involved in the ubiquitin-proteasome system. Initial fractionation of the crude reticulocyte cell extract on the anion-exchange resin diethylaminoethyl cellulose yielded two fractions which were both required to reconstitute the energy-dependent proteolytic activity that is found in the crude extract: The unadsorbed, flow
Concluding remarks
The evolution of proteolysis as a centrally important regulatory mechanism is a remarkable example for the evolution of a novel biological concept and the accompanying battles to change paradigms. The 5 decades journey between the early 1940s and early 1990s began with fierce discussions on whether cellular proteins are static as had been thought for a long time, or are turning over. The discovery of the dynamic state of proteins was followed by the discovery of the lysosome, that was
Disclosure
No relevant financial relationships with any commercial interest.
Acknowledgements
Research in the laboratory of Aaron Ciechanover is currently supported by grants from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF), the Israel Science Foundation (ISF), the I-CORE Program of the Planning and Budgeting Committee and the ISF (Grant 1775/12), and a special fund for research in the Technion established by Mr. Albert Sweet of California, USA. A.C. is an Israel Cancer Research Fund (ICRF) USA Professor. This lecture is the written version of the Nobel
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