Proteasomes. When Death Comes a Knock n. Warren Gallagher Chem412, Spring 2001

Transcription

1 Proteasomes When Death Comes a Knock n Warren Gallagher Chem412, Spring 2001

2 I. Introduction

3 Introduction The central dogma Genetic information is used to make proteins. DNA RNA Proteins Proteins are the workhorses of living cells.

4 Introduction A living sell must regulate the levels of the many proteins that are coded for by their genes. The competing processes of protein synthesis and degradation are used to control the levels of proteins in a cell. synthesis Protein degradation

5 Introduction Much is known about protein synthesis The central dogma The role of ribosomes Transcriptional and Translational control. Much less is known about protein degradation Particularly within cells

6 Introduction Over the past decade Proteasomes have emerged as one of the primary players in the intracellular degradation of proteins. Their function has been directly or indirectly linked to a wide range of diseases ranging from cancers to neurodegenerative diseases.

7 Protein Degradation Most proteins are replaced every few days The half lives, however, can vary considerably from t 1/2 =20min to t 1/2/ = days or weeks Rates can vary according to conditions Stage in cell cycle Metabolic shifts, such as starvation or viral infection

8 Protein Degradation Small proteolytic enzymes from microbial an plant sources have be exploited for years: For example Pronase Subtilisin These enzymes are used in laundry detergents contact lens cleaning solutions meat tenderizers.

9 Protein Degradation Inhibitors to some small proteases have important medical uses: HIV protease inhibitors inhibit the assembly of HIV virus particles. Angiotensin-converting enzyme inhibitors control blood pressure.

10 Protein Degradation In the past, lysosomal enzymes were thought to be primarily responsible for degrading intracellular proteins. It turns out that the lysosomes are responsible for degrading only about 10 to 20% of the intracellular proteins. Primarily those that enter the cell through phagocytosis and pinocytosis.

11 Protein Degradation The remaining 80 to 90% of intracellular proteins in eukaryotes are degraded by a large multisubunit complex called the 26S Proteasome.

12 The 26S Proteasome At 2.4 MDa, proteasomes are nearly as large as ribosomes. Approximately100 times larger than the smaller proteases. The 26S proteasome contains around 30 different protein subunits. Their activity was first discovered in the late 1970 s by Alfred Goldberg of Harvard University. They were distingueshed by their requirement for ATP. It was not until the1980 s that proteasomes were isolated by Goldberg and Rechsteiner (University of Utah), and their important role in living cells fully appreciated.

13 The 26S Proteasome In the 1990 s the structure of the 20S core portion of proteasomes was determined by X-ray diffraction and electron microscopy by Robert Huber.

14 Structure of the 26S Proteasome In eukaryotes the 26S proteasome comprises a 20S barrel-shaped core complex plus a 19S cap on either one or both ends of the 20S core.

15 Structure of the 26S Proteasome The 20S core is the site of proteolytic activity. The 19S caps are involved in regulating access to the core unfolding proteins

16 Structure of the 26S Proteasome The ATPase activity is found in the 19S caps and appears to be involved in unfolding the proteins the translocating them to the 20S core.

17 Structure of the 26S Proteasome The 20S core region in eukaryotic proteasomes is similar to structures found in bacteria and archea Bacteria Archea Eukaryote

18 Structure of the 26S Proteasome The 20S core is composed of 4 rings. The outer rings contain seven α-subunits each. the inner rings contain seven β-subunits each α β β α Bacteria Archea Eukaryote

19 Structure of the 26S Proteasome The β-subunit rings form the inner chamber where proteolysis takes place. The α-subunit rings form antechambers with the β-subunit rings and gate access to the central chamber.

20 Structure of the 26S Proteasome In archea the seven α-subunits and the seven β- subunits are usually identical. In eukaryotes each of the subunits is distinct. α β β α Chime Bacteria Archea Eukaryote

21 Function of the 26S Proteasome The catalytic activity is carried out by some (β1, β2 and β5) but not all of the β subunits. The catalytic subunits are members of the N-terminal nucleophilic hydrolase superfamily. The side chain γ-hydroxy group of Thr1 is the nucleophile The three sites have different specificitfies: Chymotrypsin (nonpolar, aromatic) Trypsin-like (polar basic) Pepidylglutramyl peptidase (glutamate) Chime

22 Function of the 26S Proteasome Proteins that are destined for destruction are covalently tagged with small protein ubiquitin. Ubiquitin is a small protein having 77 amino acids. As its name implies, it is found everywhere. Chime

23 Function of the 26S Proteasome There are three types of enzymes that are involved in ubiquitination E1 is the ubiquitin activator protein. It uses ATP to form a high energy thiolester bond between gly-76 α- carboxy group and cysteine on the E1 protein. E2 is the ubiquitin carrier protein The ubiquitin is transferred from the cysteine on E1 to a cysteine on E2. E3 is the ubiquitin ligase. E3 binds to the target protein, and then forms a ternary complex with the ubiquitinated carrier protein (E2-Ub) There are 100 s of E3 proteins E3 proteins are responsible for selecting the proteins that are to be destroyed.

24 Function of the 26S Proteasome

25 Function of the 26S Proteasome Chime

26 Function of the 26S Proteasome. Proteasome are associated with a wide range of cellular roles: Degrading foriegn proteins for peptide presentation by the MHC (major histocompatability complex) in celular immunity.

27 Function of the 26S Proteasome. Proteasome are associated with a wide range of cellular roles: Control of cell division through the timed degradation cell cycle signal proteins.

28 Function of the 26S Proteasome. Proteasome are associated with a wide range of cellular roles: Degradation of misfolded proteins.

29 Proteasome and human disease Have be directly or indirectly associated with a wide range of diseases:

30 Proteasome and human disease Have be directly or indirectly associated with a wide range of diseases: Uterine cervical carcinomas Cancer is caused by the human papilloma virus (HPV). The virus selectively targets the p53 tumor suppressor protein for degradation by the 26S proteasome It codes for an ancillory protein that associates with both the ligase (E3) and the target protein, thereby promoting ubiquination by E2-Ub.

31 Function of the 26S Proteasome Chime

32 Proteasome and human disease Have be directly or indirectly associated with a wide range of diseases: Retrovirus transformation Retroviruses code for a variant of the normal cellular c-jun transcription factor that is less susceptable to degradation by the 26S proteasome. The viral form of c_jun, called v_jun, is lacking a 27 amino acid segment in its γ domain. The lack of this domain from v_jun, a protein that is otherwise highly homologous to c_jun, provides a mechanistic explanation for the stability and resulting transforming activity of v_jun. This missing domain is not the site of ubiquination, but rather appears to serve as an anchoring site for the Jun ligase (E3)

33 Function of the 26S Proteasome Chime

34 Proteasome and human disease Have be directly or indirectly associated with a wide range of diseases: Cystic Fibrosis The mutant protein in carriers of this diseases is the 1480-aa CF transmembrane regulator (CFTR) protein, which is a regulated epithelial cell surface chloride channel protein. 70% of the known CF mutations include the deletion of phenylalanine 508. Without this phenylalanine the CFTR protein never reaches the cell surface but instead is retained in the endoplasmic reticulum where it is degraded by the 26S proteasome. Otherwise the mutant protein is physiologically capable. Inhibitors of the proteasomal activity are seen to stabilize CFTR within cells.

35 Proteasome and human disease Have be directly or indirectly associated with a wide range of diseases: Autoimmune diseases The 26S proteasome is involved in breaking down proteins for presentation by the MHC to cytotoxic T cell lymphocytes (CTL). The γ-interferon cytokine stimulate antigen presentation by inducing and exchanging three proteasomal subunits and alteration of cleavage site preference. Proteasomes appear to be non-discriminant in the peptides that they provide to the MHC Both self and non-self proteins are presented. Those from self do not normally illicit a response from the T-cells. Aberrations in the presentation process by the proteasomes may lead to the presentation of new self peptides that are treated as non-self by the T-cells.

36 Proteasome and human disease Have be directly or indirectly associated with a wide range of diseases: Neurodegenerative diseases, such as Alzheimer s, Parkinson s and Huntington s. These diseases are characterized by an accumulation of protein aggregates within neural tissues. Alteractions in the ubiquitin structure have been implicated in these diseases. Leading to ubiquitinated target proteins that are not recognized by the 26S proteasome.

37 Proteasome and human disease Have be directly or indirectly associated with a wide range of diseases: Muscle wasting Muscle wasting in individuals suffering from sepsis or starvation is tied to muscle degradation by 26S proteasomes. The peptides and amino acids that are released are used as a source of energy.

38 References D. Voges, P. Zwickl, and W. Baumeister, The 26S Proteasome: A Molecular Machine Designed for Controlled Proteolysis. Annu. Rev. Biochem. (1999) 68: A. Goldberg, S. Elledge and J.W. Harper, The Cellular Chamber of Doom, Scientific American (2001) 284: A. Schwartz and A. Ciechanover, The Ubiquitin-Proteasome Pathway and Pathogenesis of Human Diseases, Annu. Rev. Med. (1999) 50: Y. Lam et al., Inhibition of the Ubiquitin-Proteasome System in Alzheimer s Disease. Proc. Nat. Acad. Sci. USA (2000) 97: M. Gentzsh and J. Riordan, Localization of Sequences within the C-Terminal Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Which Impact Maturation and Stability. J. Biol. Chem. (2001) 276: