Intracellular protein degradation: From a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting

https://doi.org/10.1016/j.beha.2017.09.001Get rights and content

Abstract

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely non-lysosomal, but the mechanisms involved remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs.

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

References (83)

  • M. Rabinovitz et al.

    Characteristics of the inhibition of hemoglobin synthesis in rabbit reticulocytes by threo-α-amino-β-chlorobutyric acid

    Biochim Biophys Acta

    (1964)
  • A. Hershko et al.

    Mode of degradation of abnormal globin chains in rabbit reticulocytes

  • B. Poole et al.

    Some aspects of the intracellular breakdown of exogenous and endogenous proteins

  • D. Steinberg et al.

    Observations on intracellular protein catabolism studied in vitro

    Arch Biochem Biophys

    (1956)
  • A. Hershko et al.

    Studies on the degradation of tyrosine aminotransferase in hepatoma cells in culture: influence of the composition of the medium and adenosine triphosphate dependence

    J Biol Chem

    (1971)
  • A.L. Goldberg et al.

    Studies on the selectivity and mechanisms of intracellular protein degradation

  • A. Ciechanover et al.

    Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes

    J Biol Chem

    (1980)
  • K.D. Wilkinson et al.

    Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes

    J Biol Chem

    (1980)
  • T.L.K. Low et al.

    The chemistry and biology of thymosin: amino acid analysis of thymosin α1 and polypeptide β1

    J Biol Chem

    (1979)
  • L.T. Hunt et al.

    Amino-terminal sequence identity of ubiquitin and the non-histone component of nuclear protein A24

    Biochim Biophys Res Commun

    (1977)
  • A. Hershko et al.

    Identification of the active amino acid residue of the polypeptide of ATP-dependent protein breakdown

    J Biol Chem

    (1981)
  • A. Hershko et al.

    Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates

    Biochem Biophys Res Common

    (1985)
  • A. Ciechanover et al.

    N-terminal ubiquitination: more protein substrates join in

    Trends Cell Biol

    (2004)
  • M.A. Osley

    H2B ubiquitylation: the end is in sight

    Biochim Biophys Acta

    (2004)
  • A. Ciechanover et al.

    “Covalent affinity” purification of ubiquitin-activating enzyme

    J Biol Chem

    (1982)
  • A. Hershko et al.

    Components of ubiquitin-protein ligase system: resolution, affinity purification and role in protein breakdown

    J Biol Chem

    (1983)
  • A. Hershko et al.

    Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells: relationship to the breakdown of abnormal proteins

    J Biol Chem

    (1982)
  • Y. Matsumoto et al.

    Decrease in uH2A (protein A24) of a mouse temperature-sensitive mutant

    FEBS Lett

    (1983)
  • D. Finley et al.

    Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85

    Cell

    (1984)
  • A. Ciechanover et al.

    Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85

    Cell

    (1984)
  • R. Hough et al.

    Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates

    J Biol Chem

    (1986)
  • L. Waxman et al.

    Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates

    J Biol Chem

    (1987)
  • R. Hough et al.

    Purification of two high molecular weight proteases from rabbit reticulocyte lysate

    J Biol Chem

    (1987)
  • J. Driscoll et al.

    The proteasome (multicatalytic protease) is a component of the 1500-kDa proteolytic complex which degrades ubiquitin- conjugated proteins

    J Biol Chem

    (1990)
  • L. Hoffman et al.

    Multiple forms of the 20S multicatalytic and the 26S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate

    J Biol Chem

    (1992)
  • M. Hochstrasser et al.

    In vivo degradation of a transcriptional regulator: the yeast α2 repressor

    Cell

    (1990)
  • J. Adams

    Potential for proteasome inhibition in the treatment of cancer

    Drug Discov Today

    (2003)
  • H.T. Clarke

    Impressions of an organic chemist in biochemistry

    Annu Rev Biochem

    (1958)
  • R.D. Simoni et al.

    The use of isotope tracers to study intermediary metabolism: Rudolf Schoenheimer

    J Biol Chem

    (2002)
  • R. Schoenheimer

    The dynamic state of body constituents

    (1942)
  • C. de Duve et al.

    Enzymic content of the mitochondria fraction

    Nat Lond

    (1953)
  • Cited by (62)

    • Degradation-driven protein level oscillation in the yeast Saccharomyces cerevisiae

      2022, BioSystems
      Citation Excerpt :

      The output of this process is the steady-state intracellular protein concentration, maintained dynamically by continuous production and degradation (Hausser et al., 2019). The steady-state intracellular protein level shifts in response to extracellular stimuli or pathophysiologic conditions, which cause changes in production or degradation rates (Baracos 2000; Pratt et al., 2002; Mizushima and Klionsky 2007; Hinkson and Elias 2011; Ciechanover 2017; Kneppers et al., 2017). Interestingly, oscillations in the levels of key proteins have been observed in many biological phenomena (Novák and Tyson 2008), including the cell division cycle (Evans et al., 1983; Glotzer et al., 1991), circadian rhythm (Kume et al., 1999; Reppert and Weaver 2001), cellular stress response (Lahav et al., 2004; Geva-Zatorsky et al., 2006), and development (Hirata et al., 2002; Pourquié 2003).

    • The role of Siah2 in tumorigenesis and cancer therapy

      2022, Gene
      Citation Excerpt :

      Protein degradation is essential to maintain the normal physiological activity of cells. There are two sets of protein degradation pathways in human cells: one is the ubiquitin proteasome system (UPS), and the other is the lysosomal pathway (Ciechanover, 2017). Moreover, UPS disorder can lead to an abnormal protein levels of substrates, which can lead to carcinogenesis (Mansour, 2018).

    • Intracellular peptides as drug prototypes

      2022, Peptide and Peptidomimetic Therapeutics: From Bench to Bedside
    • Potential roles of natural products in the targeting of proteinopathic neurodegenerative diseases

      2021, Neurochemistry International
      Citation Excerpt :

      Chaperons are classified as small heat-shock proteins that interact hydrophobically with misfolded proteins to prevent aggregations (Haslbeck et al., 2005) and refold protein with the aid of HSP70 chaperones, which are also involved in protein disaggregation and proteolytic degradation (Kettern et al., 2010). The protein degradation system includes autophagy and ubiquitin-proteasome degradation (Boland et al., 2018; Ciechanover, 2017; Dikic, 2017; Vendruscolo et al., 2011), which are referred to as protein clearance systems, and these systems remove misfolded proteins to ensure proteostasis (Boland et al., 2018). Hipp et al. found that misfolded proteins, caused by mutations, translation errors, or OS, conjugate better with chaperones and are more likely to be degraded by the ubiquitin-proteasome system (UPS) (Hipp et al., 2014b).

    View all citing articles on Scopus
    View full text