`Molecular Biochemistry II

Protein Degradation

Contents of this page:
Serine proteases
Other classes of proteases
Lysosomes & protein turnover
Ubiquitin
Proteasome core complex
Regulatory cap complexes

Classes of proteolytic enzymes:

Serine proteases include the digestive enzymes trypsin, chymotrypsin, and elastase. Different serine proteases differ in substrate specificity. For example, chymotrypsin prefers an aromatic side chain on the residue whose carbonyl carbon is part of the peptide bond to be cleaved (R-group colored blue below). Trypsin prefers a positively charged Lys or Arg residue at this position.

In the diagram above a small peptide is shown being cleaved, while the usual substrate would be a larger polypeptide. The active site in each serine protease includes a serine residue, a histidine residue, and an aspartate residue. During attack of the serine hydroxyl oxygen, a proton is transferred from the serine hydroxyl to the imidazole ring of the histidine, as the adjacent aspartate carboxyl is H-bonded to the histidine.

Aspartate proteases include the digestive enzyme pepsin, some proteases found in lysosomes, the kidney enzyme renin, and the HIV-protease. Two aspartate residues participate in acid/base catalysis at the active site. In the initial reaction, one aspartate accepts a proton from an active site H2O, which attacks the carbonyl carbon of the peptide linkage. Simultaneously, the other aspartate donates a proton to the oxygen of the peptide carbonyl group.

Cysteine proteases have a catalytic mechanism that involves a cysteine sulfhydryl group. Deprotonation of the cysteine sulfhydryl by an adjacent histidine residue is followed by nucleophilic attack of the cysteine S on the peptide carbonyl carbon. A thioester linking the new carboxy-terminus to the cysteine thiol is an intermediate of the reaction (comparable to the acyl-enzyme intermediate of a serine protease). Cysteine proteases include the following:

• Papain is a well-studied plant cysteine protease.
• Cathepsins are a large family of lysosomal cysteine proteases, with varied substrate specificities.
• Caspases are cysteine proteases involved in the activation and implementation of apoptosis (programmed cell death). Caspases get their name from the fact that they cleave on the carboxyl side of an aspartate residue.
• Calpains are Ca⁺⁺-activated cysteine proteases that cleave intracellular proteins involved in cell motility and adhesion. They regulate processes such as cell migration and wound healing.

Activation of proteases:

• Most proteases are synthesized as larger pre-proteins. During activation, the pre-protein is cleaved to remove an inhibitory segment.
• In some cases activation involves dissociation of an inhibitory protein (see below).
• Activation may occur after a protease is delivered to a particular compartment within a cell or to the extracellular milieu. 
• Caspases involved in initiation of apoptosis are activated by interaction with large complexes of scaffolding and activating proteins called apoptosomes. For a diagram of an apopotosome complex see a University of London website.

Protease inhibitors: Most protease inhibitors are proteins with domains that enter or block a protease active site to prevent substrate access.

• IAPs are proteins that block apoptosis by binding to and inhibiting caspases. The apoptosis-stimulating protein Smac antagonizes the effect of IAPs on caspases.
• TIMPs are inhibitors of metalloproteases that are secreted by cells. A domain of the inhibitor protein interacts with the catalytic Zn⁺⁺. 
• Cystatins are inhibitors of lysosomal cathepsins. Some of these (also called stefins) are found in the cytosol and others in the extracellular space. Cystatins protect cells against cathepsins that may escape from lysosomes. 
• Serpins are widely distributed proteins that utilize a unique suicide mechanism to inhibit serine or cysteine proteases.
  A large conformational change in the serpin accompanies cleavage of its substrate loop. This leads to disordering of the protease active site, preventing completion of the reaction. The serpin remains covalently linked to the protease as an acyl-enzyme intermediate.
  See the movie "Protease Inhibition" in the Structural Medicine website, University of Cambridge.
  Serpins are widely distributed within & outside of cells, and have diverse roles, including regulation of blood clotting, fibrin cleavage, and inhibition of apoptosis.

Lysosomes contain a large variety of hydrolytic enzymes that degrade proteins and other substances taken in by endocytosis. Materials taken into a cell by inward budding of vesicles from the plasma membrane may be processed first in an endosomal compartment and then delivered into the lumen of a lysosome by fusion of a transport vesicle. For a brief introduction to receptor-mediated endocytosis, see class materials on lipoproteins. Solute transporters embedded in the lysosomal membrane catalyze exit of products of lysosomal digestion (e.g., amino acids, sugars, cholesterol) to the cytosol. Lysosomes have a low internal pH due to activity of vacuolar ATPase, a H+ pump homologous to (but distinct from) the mitochondrial F1Fo ATPase. All intra-lysosomal hydrolases exhibit acidic pH optima.  Lysosomal proteases include many cathepsins (cysteine proteases), as well as some aspartate proteases and one zinc protease. Activation of lysosomal proteases by cleavage may be catalyzed by other lysosomal enzymes or be autocatalytic, promoted by the acidic pH within the lysosome.

Protein turnover and selective degradation or cleavage:

Individual cellular proteins turn over (are degraded and re-synthesized) at different rates. For example, half-lives of selected enzymes of rat liver cells range from 0.2 to 150 hours (Table p. 1352).

N-end rule: On average, a protein's half-life correlates with its N-terminal residue. See table p. 1357.

• Proteins with N-terminal Met, Ser, Ala, Thr, Val, or Gly have half lives greater than 20 hours.
• Proteins with N-terminal Phe, Leu, Asp, Lys, or Arg have half lives of 3 min or less.

PEST proteins, rich in Pro (P), Glu (E), Ser (S) and Thr (T), are more rapidly degraded than other proteins.

Most autophagy is not a mechanism for selective degradation of individual macromolecules. However, cytosolic proteins that include the sequence KFERQ may be selectively taken up by lysosomes in a process called chaperone-mediated autophagy. This process, which is stimulated under conditions of nutritional or oxidative stress, involves interaction of proteins to be degraded with:

• Cytosolic chaperones that unfold the proteins.
• A lysosomal membrane receptor (LAMP-2A) that may provide a pathway across the membrane.
• Chaperones in the lysosomal lumen that may assist with translocation across the membrane.

Intramembrane-cleaving proteases (I-CLiPs) cleave regulatory proteins such as transcription factors from membrane-anchored precursor proteins. For example, precursors of SREBP (sterol response element binding protein) transcription factors are integral proteins embedded in endoplasmic reticulum membranes. Activation of SREBP involves its translocation to golgi membranes where sequential cleavage by two proteases releases to the cytosol a domain with transcription factor activity. The released SREBP can then translocate to the cell nucleus to regulate transcription of genes for enzymes involved, e.g., in cholesterol synthesis. S2P (site 2 protease, an I-CLiP) is a membrane-embedded metalloprotease that cleaves an a-helix of the SREBP precursor within the transmembrane domain.

An isopeptide bond links the terminal carboxyl of ubiquitin to the e-amino group of a lysine residue of a "condemned" protein. The joining of ubiquitin to a condemned protein is ATP-dependent. Three enzymes are involved, designated E1, E2 and E3. (See also p. 1354.) Initially, the terminal carboxyl group of ubiquitin is joined in a thioester bond to a cysteine residue on Ubiquitin-Activating Enzyme (E1). This is the ATP-dependent step.

The ubiquitin is then transferred to a sulfhydryl group on a Ubiquitin-Conjugating Enzyme (E2). A Ubiquitin-Protein Ligase (E3) then promotes transfer of ubiquitin from E2 to the e-amino group of  a lysine residue of a protein recognized by that E3, forming an isopeptide bond.  There are many distinct Ubiquitin Ligases with differing substrate specificity.  One E3 is responsible for the N-end rule. Some E3s are specific for particular proteins. Some proteins (e.g., mitotic cyclins involved in regulation of the cell cycle) have a sequence called a destruction box that is recognized by a domain of the corresponding Ubiquitin Ligase.

Ubiquitin Ligases (E3) mostly consist of two families.

• Some Ubiquitin Ligases have a HECT domain containing a conserved cysteine residue that participates in transfer of activated ubiquitin from E2 to a target protein.
• Some Ubiquitin Ligases contain a RING finger domain in which cysteine and histidine residues are ligands to 2 Zn⁺⁺ ions. A RING (Really Interesting New Gene) finger is not inherently catalytic. It stabilizes a characteristic globular domain conformation that serves as a molecular scaffold for residues that interact with E2.

Regulation of ubiquitination:

Proteins have been identified that regulate or facilitate ubiquitin conjugation. Regulation by phosphorylation of some target proteins has been observed.  For example, phosphorylation of PEST domains activates ubiquitination of proteins rich in the PEST amino acids. Glycosylation of some PEST proteins with N-acetylglucosamine (GlcNAc) has the opposite effect, prolonging the half-life of these proteins. GlcNAc attachment increases with elevated extra-cellular glucose, suggesting a role as a nutrition sensor.

• A ubiquitin-like protein called Nedd8 may be attached to ubiquitin ligases (E3) that have a "cullin" subunit including a RING finger domain. De-neddylation (removal of the Nedd8 protein), catalyzed by a metalloprotease subunit of a complex called the COP9 signalosome, activates the E3 ligases.
• Some disease-causing viruses target host cell proteins for degradation in the proteasome. They either activate a host cell ubiquitin ligase to ubiquitinate host proteins, or encode their own Ubiquitin Ligase.

Proteasomes: Selective protein degradation occurs in the proteasome, a large protein complex located in the nucleus and cytosol of eukaryotic cells. The proteasome core complex, which has a sedimentation coefficient of 20S, contains 2 copies each of 14 different polypeptides. 7 a-type proteins form each of the two a rings, at the ends of the cylindrical structure. 7 b-type proteins form each of the two central b rings. The 20S proteasome core complex encloses a cavity consisting of 3 compartments joined by narrow passageways. See diagrams p. 1360, 1362.

Protease activities are associated with three of the b subunits, each having different substrate specificity:

• One catalytic b subunit has a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 (peptide cabonyl) position.
• One has a trypsin-like activity with preference for arginine or lysine at the P1 position.
• One has a post-glutamyl activity with preference for glutamate or other acidic residue at the P1 position.

Different variants of the three catalytic subunits, with different substrate specificity, are produced in cells of the immune system that cleave proteins for antigen display.

The proteasome hydrolases constitute a unique family of threonine proteases. A conserved N-terminal threonine is involved in catalysis at each active site. The three catalytic b subunits are synthesized as pre-proteins. They are activated when the N-terminus is cleaved off, making threonine the N-terminal residue. Catalytic threonines are exposed at the lumenal surface.

Proteasomal degradation of particular proteins is an essential mechanism by which cellular processes are regulated, such as cell division, apoptosis, differentiation and development. For example, progression through the cell cycle is controlled in part through regulated degradation of proteins called cyclins that activate cyclin-dependent kinases.

Many inhibitors of proteasome protease activity are known, some of which are natural products and others experimentally produced. E.g., TMCs are naturally occurring proteasome inhibitors. They bind with high affinity adjacent to active site threonines within the proteasome core complex. TMCs have a heterocyclic ring structure derived from modified amino acids.

Proteasome inhibitors cause cell cycle arrest and induction of apoptosis (programmed cell death) when added to rapidly dividing cells. The potential use of proteasome inhibitors in treating cancer is being investigated.

Several subunits of the proteasome are glycosylated with GlcNAc (N-acetylglucosamine) when extracellular glucose is high, leading to decreased intracellular proteolysis. Conversely, under conditions of low nutrition, decreased modification by GlcNAc leads to increased proteolysis. Thus protein degradation is responsive to nutrition via glycosylation of both Ubiquitin Ligase (see above) and the proteasome itself.

Regulatory cap complexes: In crystal structures of the proteasome core complex alone, there is no apparent opening to the outside. The ends of the cylindrical complex are blocked by N-terminal domains of a subunits that function as a gate. Interaction with a cap complex causes a conformational change that opens a passageway into the core complex.

The 19S regulatory cap complex recognizes multi-ubiquitinated proteins, unfolds them, removes ubiquitin chains, and provides a passageway for threading unfolded proteins into the proteasome core complex. The 19S cap is a 20-subunit 700 kDa complex, also referred to as PA700. When combined with a 20S core complex, it yields a 26S proteasome. Only low-resolution structural information, obtained by electron microscopy, is available for the 19S cap (see p.1355). Location and roles of some constituent proteins have been established.

• The outermost "lid" of the 19S cap consists of a ring of eight proteins.
• The innermost "base" of the 19S cap includes a ring of six members of the AAA family of ATPases. These are chaperones that carry out ATP-dependent unfolding of proteins prior to their being threaded into the core complex. It is typical of AAA ATPases that they assemble into hexameric rings.
• Isopeptidases in the 19S cap disassemble ubiquitin chains. Ubiquitins can then be re-used. At least one deubiquitylating enzyme is known to be located between the lid and base regions of the 19S cap.

A simpler archaebacterial cap complex called PAN consists only of a hexameric ring of AAA ATPases, comparable to the base of the 19S regulatory cap. PAN, in the presence of ATP, was found to cause opening of a gate at the end of the 20S proteasome through which an unfolded protein could enter. The base of the19S cap is assumed to do the same, although high resolution structural evidence for this is still lacking.

The 11S regulatory cap is a heptameric complex of a protein PA28. It allows small, non-ubiquitinated proteins and peptides to pass into the proteasome core complex. This does not require ATP hydrolysis. The 11S cap is dome-shaped, with a wide opening at each end. Binding of the 11S cap alters the conformation of N-terminal domains of core complex a subunits, opening a gate into the proteasome core. For images showing conformational changes involved in gate opening by the 11S cap see the website of Christopher Hill. There have been many structural studies of isolated proteasome core complex with either the 19S or 11S cap (as at right). Formation of mixed complexes, in which a proteasome core is sandwiched between 19S and 11S caps, has been demonstrated by electron microscopy. In vivo, a 19S cap may recognize, de-ubiquitinate, unfold and feed proteins into a core complex at one end, while an 11S cap at the other end may provide an exit path for peptide products.

View at right an animation depicting degradation of a ubiquitinated protein in the proteasome. Copyright © 1998-2008 by Joyce J. Diwan. All rights reserved.

of protein degradation in the proteasome

Additional material on Protein Degradation: Readings, Test Questions & Tutorial