Proteolysis into the next Millennium

(A contribution made in 2000 by Professor R John Mayer, Professor of Molecular Cell Biology, University of Nottingham, United Kingdom, amended and edited by Dr Paul W Sheppard, Scientific Development Director, AFFINITI Research Products Ltd, Exeter, United Kingdom.)

1. Reflections - a waste of time and energy!

The notion that intracellular proteolysis would be of physiological significance was widely discounted for many years because degrading proteins after synthesis would be "energetically-wasteful". Furthermore, the notion that the destruction of specific proteins might be a key step in the physiological regulation of signal transduction pathways was also refuted: it would be necessary to make the protein again. Prejudice in science is curious: both premises are wrong. Proteins are degraded continuously and non-continuously as directed by physiological need.

Intracellular proteolysis has lysosomal and non-lysosomal components: the latter being mediated predominantly by the ubiquitin/26S proteasome system. However, inter-relationships might be expected between the systems and are now starting to become apparent.

2. Intracellular proteolysis - the ultimate regulator of proteomic function?

(i) cell cycle: the magic roundabout?

The biochemical mechanisms that regulate the cell cycle are the subject of intense investigation. The G1/S, G2/M and mitotic phases of the cell cycle are controlled by the actions of cyclin-dependent kinases, kinase inhibitors, phosphatases and ubiquitin/26S proteasome-dependent proteolysis. Critical phosphorylation events attract ubiquitin-protein ligases which ubiquitinylate cyclins and kinase inhibitors in preparation for degradation by the 26S proteasome. The SCF family of ubiquitin-protein ligases may be responsible for protein ubiquitinylation in the G1/S phase and the related APC/cyclosome complexes perform the same function in G2/M. The search continues for further substrates that are targeted for degradation during the cell cycle and cytokinesis (King et al., 1996). Evidence is beginning to accumulate that protein delivery to the 26S proteasome may require more than substrate protein multi-ubiquitinylation. Accessory or adaptor molecules may be involved in protein binding to the 19S regulator of the 26S proteasome (Higashitsuji et al., 1999).

 (ii) transcription: complexes and complexity

Transcription factors are lethal molecules: too much or too little of these controllers of gene expression has catastrophic consequences for the cell.

Transcription factors are degraded by the ubiquitin/26S proteasome system. For example, the increased expression of hypoxia-sensitive genes is controlled by hypoxia-inducible transcription factors (HIF). HIF are degraded by the ubiquitin/26S proteasome system. Amongst the HIF-controlled genes are those responsible for angiogenesis. In von Hippel-Lindau (VHL) disease, kidney tumours are associated with increased angiogenesis. Mutations in the VHL tumour suppressor can cause these tumours. The VHL protein is a component of a VHL-elonginB/C/cullin2 complex similar to SCF ubiquitin-protein ligase (Kaelin and Mather, 1998).

In colonic cancer, mutations in the adenomatous polyposis coli (APC) protein occur. The APC protein is part of a kinase complex that phosphorylates proteins before ubiquitinylation (Behrens et al., 1998).

(iii) signal transduction: pathway terminators

Ubiquitinylation of the lymphocyte homing receptor and other membrane receptors including growth-hormone receptor and the platelet-derived growth factor receptor has been known for many years. More recently, downstream adaptor proteins for membrane receptor proteins have been shown to be ubiquitinylated, e.g. cCbl. Receptor down-regulation by ubiquitin-dependent degradation is an important aspect of signal transduction. Receptor ubiquitinylation is a complex process with mono-ubiquitinylation acting as a internalisation signal as well as multi-ubiquitinylation serving as the degradation signal (Hicke, 1999; Terrell et al., 1999). Again, as in cell cycle control, it appears that kinases, phosphatases and ubiquitin-dependent proteolysis control key cell physiological processes.

 (iv) antigen processing: bits and pieces

Although not absolutely exclusively, proteasomes control protein fragmentation as part of MHC Class I antigen processing. Proteins are broken down into small peptides (9-13 amino acids) which move into the endoplasmic reticulum (ER) where they can bind to MHC molecules and trigger export of peptide-MHC complexes to the cell surface to activate cytotoxic lymphocytes. Furthermore, interferon-g can cause cells to incorporate new catalytic subunits into new 20S proteasomes with an increased ability to generate protein fragments to trigger a better Class I response and also cause the expression of subunits of the 11S regulator of the 20S proteasome which, again, facilitates the production of protein fragments which bind optimally to MHC Class I molecules and accelerate the cytotoxic lymphocyte response (Groettrup et al., 1997).

 (v) destruction alternatives: dump or larder?

Chronic human neurodegenerative diseases are associated with the formation of perinuclear protein aggregates (inclusions) in neurones (Mayer et al., 1999). Similarly, during some latent viral infections, e.g. Epstein Barr Virus (EBV), latent membrane protein (LMP) accumulates in EBV-transformed lymphoblastoid cells in pericentriolar inclusions (Laszlo et al., 1991).

Recently, it has been shown that both the mutant cystic fibrosis transmembrane regulator (CFTR) and mutant presenilin-1 accumulate in "aggresomes" in the pericentriolar region (Johnston et al., 1998; Wigley et al., 1999). These aggresomes are enriched in components of the ubiquitin/26S proteasome pathway and cell stress proteins. Additionally, a SCF ubiquitin-protein ligase has a rôle in centrosome duplication (Freed et al., 1999). Maybe, the ubiquitinylation/26S proteasomal apparatus is focussed on the pericentriolar region not only to facilitate cell division but also to ubiquitinylate mutant proteins (and excess wild-type proteins?) which have been removed from the ER by the ER-quality control system (Hiller et al., 1996). However, instead of being degraded, excised ubiquitinylated proteins are deposited in the pericentriolar region either simply to prevent toxic gains of function of the ubiquitinylated proteins or for subsequent degradation by the 26S proteasome system or the lysosomal system (Doherty et al., 1987; Earl et al., 1987).

 3.  The ubiquitinylation machinery and the proteases - nuts and bolts

(i) ubiquitons: variations on a superfold

There are cellular proteins related to ubiquitin but differing in terms of primary sequence and three-dimensional structure. Furthermore, these "ubiquitons" are either free and conjugatable or are genetically built into proteins, e.g. RAD23 and Parkin. The key element in these molecules is the ubiquitin-superfold and the utilisation of this superfold for a variety of purposes during protein-protein interactions (Mayer et al., 1998). The attachable SUMO/Smt3p/ Sentrin/Pic1/Gmp1 and NEDD8/Rub1 ubiquitons have roles in protein-import into the nucleus (Matunis et al., 1996) and the regulation of ubiquitin-protein ligase activity, respectively (Liakopoulos et al., 1998). The functions of built-in ubiquitons are less obvious, although RAD23 has a role in nucleotide excision repair and binds to the so-called ubiquitin binding subunit (S5a) of the 19S regulator of the 26S proteasome (Hiyami et al., 1999).

 (ii) autophagy: squaring the circle

The basis of the utilisation of ubiquitin (and attachable ubiquitons) is the formation of an isopeptide bond between the carboxylic acid moiety of the carboxyl-terminal glycine residue of ubiquitin and the e-amino group of a lysine residue within a target protein. Remarkably, the evolution of the enzymology to generate isopeptide bonds, which link one protein to another, might be the "commonality" in intracellular proteolysis.

Recently, an isopeptide system has been discovered which links proteins together as part of the mechanism of autophagolysosome formation (Mizushima et al., 1998a, 1998b; Shintani et al., 1999). Genes coding for enzymes with analogous functions to a ubiquitin activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2) have been discovered which control early events in autophagy in yeast and in man. Given the diversity of proteins containing genetically built-in ubiquitons, it is predictable that the autophagic regulator protein in yeast (apg12p) which is conjugated to another protein (apg5p) by an isopeptide-bond will have a ubiquitin superfold: time and crystallography will tell!

 (iii) ubiquitin-protein ligases: ultimate arbiters

"To be or not to be" may be part of the question: however, ubiquitin-protein ligases are the answer!

Currently, there are two families of ubiquitin-protein ligases: the HECT domain (homologous to the E6-accessory protein [AP] carboxyl-terminus) enzymes (which form thioesters with ubiquitin) and the RING finger ligases. The E6 protein is encoded in malignant forms of papilloma viruses and through recruitment of the cellular E6-AP ubiquitin-protein ligase causes the degradation of p53. The RING finger proteins are either in complexes with other proteins essential for ligase activity (the SCF and APC/cyclosome complexes) or are associated with putative substrate proteins. The RING finger ubiquitin-protein ligases bind ubiquitin-conjugating enzymes to facilitate ubiquitinylation of target proteins. The latter group of RING finger ligases includes c-Cbl, which is an adaptor for receptor protein tyrosine kinases. The c-Cbl protein binds to phosphorylated tyrosine residues in activated receptors via SH2 domains and triggers ubiquitinylation via the associated ubiquitin-conjugating enzyme (Joazeiro et al., 1999). Other RING finger proteins may act as ubiquitin-protein ligases. For example, the protein product of the breast cancer 1 gene (BRCA1) has a RING finger and is a ubiquitin-protein ligase (Lorick et al., 1999). At least 7 other RING finger proteins have demonstrated ubiquitin-protein ligase activity.

There are over 400 proteins with RING fingers in the database. These proteins could be bone fide ubiquitin-protein ligases: there are certainly sufficient substrates amongst the products of the 70,000-100,000 human genes that code for proteins (O'Brien et al., 1999)!  Alternatively, evolution may have contrived a system whereby RING finger proteins remain inactive in functional complexes, e.g. c-Cbl, until activated, e.g. in response to receptor tyrosine kinase ligands, when the RING finger recruits a ubiquitin-conjugating enzyme. This ubiquitinylates either the RING finger protein or some putative protein substrate in the functional complex (Lorick et al., 1999). Either way, the ubiquitin-conjugating enzyme becomes active.

 (iv) non-lysine48-linked ubiquitin chains: which and why?

Ubiquitin molecules, which are linked together in chains to a protein as a degradation signal, are covalently coupled via an isopeptide bond as described earlier utilising the lysine⁴⁸ (K⁴⁸) residue of each ubiquitin. However, chains have also been shown to be linked via four of the other six lysines in ubiquitin (K⁶, K¹¹, K²⁹, and K⁶³). The K⁶³-linked polyubiquitin chains appear to play a role in DNA repair. The formation of K⁶³-linked chains is not a signal for degradation, which means that attachment of K⁶³-linked chains to proteins (if this is a widespread process?) is not for degradation but for some other purpose, probably in the nucleus in DNA repair. The generation of K⁶³-linked chains is through a heterodimer composed of an ubiquitin-conjugating enzyme variant (UEV) and a specific ubiquitin-conjugating enzyme, ubp13p (Hofmann and Pickart, 1999). The UEV proteins are homologous to ubiquitin-conjugating enzymes but lack the critical catalytic cysteine residue. The UEV proteins have been implicated in cell transformation and tumour suppression. Again a protein, the ubiquitin-conjugating enzyme variant, activates an ubiquitin-conjugating enzyme. How often will ubiquitin-conjugating enzymes be found to be complexed to other proteins in order to be active?

 (v) proteasome interaction partners: friends and foes

Many cellular and viral proteins have been shown to interact with both 20S and 19S proteasomal subunits. The six non-redundant ATPases of the 19S regulator, which sit as a "collar" (base) along with two non-ATPases on each end of the 26S proteasome, have been shown to interact with many cellular and viral proteins. Presumably, these interactions are to modulate the recognition/degradation of either the binding proteins themselves or other cellular and viral proteins. For example, the HEC protein (highly expressed in cancer) specifically interacts with the S7 ATPase and modulates the degradation of a mitotic cyclin (Chen et al., 1997), whilst the papilloma virus E7 protein specifically interacts with the S4 ATPase and controls the degradation of the retinoblastoma protein (Boyer et al., 1996; Berezutskaya and Bagchi, 1997). Recently, a cellular protein, gankyrin, which interacts with the S6 ATPase, has been discovered to be an oncoprotein which increases the degradation of the retinoblastoma protein (Higashitsuji et al., 1999). Perhaps, E7 mimics gankyrin?

 (vi) proteasome assembly: THE Millennium structure

The assembly of the 20S core of the 26S proteasome has been solved in part by studies in Thermoplasma (Lupas et al., 1997) and other organisms (Schmidtke et al., 1996). However, the details of the assembly of the heptameric a-rings and the role of the heptameric b-rings in 20S particle formation has yet to be fully resolved. Some a-subunits, e.g. a7 (C8) may have co-ordinating roles for the assembly of the heptameric a-rings (Gerards et al., 1998). The mode of assembly of the 19S regulators of the 26S proteasome is poorly understood, although the elegant demonstration that the 19S regulator consists of a "base" containing the six ATPases and two other proteins, with the other regulatory proteins in the "lid", helps to clarify the basic structural features of the 19S complex on which to build details of the assembly process (Glickman et al., 1998).

(vii) deubiquitinating enzymes: more yin than yang

The sequencing of the yeast genome has revealed that there are more genes coding for deubiquitinating enzymes than ubiquitin-conjugating enzymes; the precise functions of these enzymes are unknown but several of the enzymes are functionally non-redundant. Deubiquitinating enzymes are crucial for cellular proteolysis including ubiquitin-chain disassembly (Amerik et al., 1997) and ubiquitin chain-editing by the 26S proteasome (Lam et al., 1997). Deubiquitinating enzymes have key roles in cell cycle regulation (Zhu et al., 1996, 1997) and interact with the BRCA1 protein (Jensen et al., 1998), which is one of the RING finger ubiquitin-protein ligases (Lorick et al., 1999), perhaps in some large complex involving DNA repair and protein ubiquitinylation (see iii above) and deubiquitinylation. Clearly, these enzymes will have important roles in cellular physiology in addition to the ubiquitin-conjugating pathway.

 (viii) tripeptidyl peptidases: back to basics

The ubiquitin/26S proteasome system can degrade proteins to small peptides. There must be one or more enzyme systems that can then produce the basic building blocks of proteins, the amino acids, from such peptides. One strong candidate is tripeptidyl peptidase, also a megaprotein complex, capable of cleaving a variety of peptides into tripeptides for further excision into amino acids by exopeptidases (Tomkinson, 1999). Other enzymes may assist in the total degradation of proteins into amino acids and their full characterisation is keenly awaited.

 (ix) substitutes: key players in the game

Evolution enjoys compensation by substitution. Clones of cells deprived of the activity of 20S proteasomes by inhibition with, for example, lactacystin can survive by using another protease to degrade proteins and process antigens (Glas et al., 1998). The compensatory protease may be the gigantic compartmentalised tricorn protease (Tamura et al., 1998). The full significance of this protease complex is yet to be determined.

 4. Prospects - hear today and here tomorrow

Intracellular proteolysis is the most recently discovered regulatory system of cellular physiology. The field has undergone a Cinderella-like "rags to riches" growth in the last five years. Predictably, within the next five years, driven by genomics and proteomics, not to mention good old-fashioned biochemistry - would that be functional genomics?! - everything from cell division, development and differentiation to cellular senescence will be found to have a proteolytic component. There is no simpler way to stop a physiological process than to destroy one of the components of a pathway in a controlled fashion. Already, a keen interplay between phosphorylation, ubiquitinylation and degradation can be seen (Yaron et al., 1997). Ubiquitinylation will support and rival phosphorylation in the regulation of the life process. After all, what is the conceptual difference between the addition and removal of phosphates or acyl-groups? It now appears that acetylation controls transcription (Brehm et al., 1999) whilst ubiquitinylation not only contributes to the control of transcription but modulates a host of other cell physiological processes as well.

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