Download Protein Degradation As discussed in last the last lecture, newly

Protein Degradation
As discussed in last the last lecture, newly synthesized proteins are folded and
glycosylated in the ER in preparation for their transport to other organelles. However,
life is not always perfect in the ER: proteins may turn out to have mutations that prevent
proper folding, or poisonous compounds may inhibit glycosylation. Similarly, some of
the proteins in the cytoplasm may also be misfolded, or damaged by oxygen radicals or
other compounds, and aggregate. If all this junk is allowed to accumulate, the cell would
die. Therefore, the cell rapidly breaks such protein down. Hence a need for a proteolysis
pathway. In addition to removal of misfolded proteins, proteolysis is also important in
recycling or turnover of proteins and amino acids, as well as regulation of processes such
as cell cycle, transcription, cell signaling, and apoptosis.
Proteolysis takes place primarily in the cytoplasm. So why don’t “normal” cytosolic
proteins get chewed up? The first reason is that protein degradation occurs in defined
compartments that are excluded from the cytosol. Secondly, proteins that are destined to
be degraded are often marked. We’ll look at the organelles or machines involved in
protein degradation, and then see how ER proteins, which are excluded from the cytosol,
get degraded.
I. Lysosomes. The lysosome is a membrane bound organelle that contains enzymes
involved in the degradation of a variety of substances. ( MBOC p. 610 Fig. 13-17)
Lysosomes are the main organelle for the degradation of extracellular materials, which
are collected by endocytosis, which will be discussed in detail in later lectures.
Lysosomes also digest entire organelles or parts of the cytoplasm that are engulfed in a
process known as autophagy. ( MBOC p. 614 Fig. 13-22) There is yet a third, more
specific process. During stress situations such as starvation, there is a large turnover of
proteins in the cell. Studies have shown that RNase A, and other cytosolic proteins
bearing the amino acids sequence KFERQ, are directly and selectively transported to the
lysosome for degradation (V&V p. 1011).
About 40-50 acid hydrolases reside in the lysosome. In addition to proteases, there are
nucleases, glycosylases, lipases, phospholipases etc. They are acid hydrolases because
their optimal pH for activity is about 5, which is the pH inside lysosomes. The cell is
smart in that if a lysosome accidentally ruptures and releases its contents to the cytosol,
the cell won’t be chewed up, because the enzymes will not be active.
If you isolate lysosomes, and put them in a test tube with solution that has ADP and Pi,
ATP is made and the pH of the solution goes up. What does this tell you? As you could
guess by now, the internal pH is maintained by a proton pump that derives its energy
through ATP hydrolysis. This pump is formally known as the vesicular ATPase, or VATPase.
A variety of proteases exists inside the lysosome (V&V p. 1011). These include
carboxypeptidase, aminopeptidase, and endopeptidase. Most of them belong to the class
known as cathepsins. Many of these are cysteine proteases. Like the serine proteases,
they also have a catalytic triad, this time consisting of Cys-His-Asn.
II. The Ubiquitin-Proteasome Pathway
This is the major pathway by which cytosolic proteins are selectively degraded during
normal metabolic growth. As mentioned, it is also very important for regulation of
processes such as cell cycle control and cell signaling.
A. Ubiquitination
The first step is the attachment of a signal/flag to the protein that is to be degraded. In
this case the signal is a polyubiquitin chain. Ubiquitin is a 72 amino acids polypeptide
that is very abundant, as its name suggests. Linkage of ubiquitin to the “condemned”
protein occurs in 3 enzyme-catalyzed steps (see V&V p. 1012 Fig. 30-59).
1. E1- ubiquitin – a thioester bond is formed between the C-terminal amino acids of
ubiquitin and an internal Cys residue of E1, the ubiquitin activating enzyme. This is
an ATP dependent process.
2. E2- ubiquitin – an exchange reaction occurs, and the ubiquitin is transferred to a
sulfhydryl group on E2, the ubiquitin-conjugating enzyme. The thioester bond is
preserved and no ATP is required in this reaction.
3. E2-ubiquitin then interacts with E3, to which the substrate is specifically bound. E3 is
known as the ubiquitin-protein ligase. It transfers the activated ubiquitin to the ε-NH2
group on a lysine residue in the protein substrate, thus establishing an amide linkage.
In subsequent reactions, additional ubiquitin molecules are added to an ε-NH2 group
on a lysine residue of the previously conjugated ubiquitin molecule to form a multiubiquitin chain linked by isopeptide bonds.
B. The specificity of the system is conferred primarily by E3. To date there are 4 main
classes that have been identified (refer to the handout on protein degradation).
1. N-end rule E3s. The residue at the N-terminus of proteins affect their half-lives (see
V&V p. 1014 and MBOC p. 220).
E3α is one of the E3s that have been identified. It has two distinct sites that
recognize either basic (type1) or bulky-hydrophobic (type 2) residues. It also
recognizes other non-N-end rule substrates via its “body site”.
2. HECT-domain E3s. (homologous to the E6-AP C-terminus). E6-AP is a cellular
protein that forms a complex with the human papillomavirus protein E6 (thus
AP=associated protein). Together they act like an E3 to target p53, a tumor
suppressor protein in the cell, for degradation. The conserved HECT domain is in the
C-terminus of these E3s, and has catalytic function, and an invariable Cys residue.
The N-terminus of HECT-domain E3s is not highly conserved. Rsp5 is an example
of a HECT-domain E3. It contains a calcium-binding domain, C2, and 3 WW
domains in the amino-terminal two-thirds of the protein. The WW domains are in
part responsible for substrate recognition.
3. Cyclosome/APC (anaphase promoting complex). Cyclosomes are inactive during
interphase. They are activated by a mitotic kinase. When phosphorylated, they
specifically recognize cell-cycle regulators such as mitotic cyclins that contain a 9
amino acids motif termed the destruction box. While N-end rule and HECT-domain
E3s are single polypeptides, the cyclosome has several subunits. Cells that have
mutations in any of these subunits fail to degrade B-type cyclins, cannot complete or
exit from mitosis.
4. Phosphoprotein-ubiquitin ligase complexes. These complexes also degrade cell cycle
regulators. However, their substrates need to be phosphorylated before these E3s will
recognize them. While certain subunits in these complexes are invariant, others can
be swapped to alter the substrate specificity.
C. Proteasome
Structure (see MBOC p. 219 Fig. 5-37): The core of the proteasome is a 20S complex
composed of 4 stacked rings. There are 2βrings on the inside and 2 α rings on the
outside. Each ring is composed of 7 subunits. A central channel is present in the 20S
complex. This arrangement is reminiscent of GroEL. The opening at the top created by
the alpha subunits is about 13 Å and is just big enough for an unfolded chain to enter.
The 19S Cap complex normally sits on top of the alpha ring, rather like GroES. The 19S
cap consists of more than 15 subunits, including ATPases. The functions of the 19S
complex includes recognition of condemned proteins via the ubiquitin chain (another way
to prevent the accidental degradation of cytoplasmic proteins), release and subsequent
hydrolysis of the ubiquitin chain, unfolding of the condemned polypeptide, and
translocation of the unfolded protein into 20S. It may also control the exit of degradation
products. The step(s) that require energy has not been clearly demonstrated, though it is
highly likely that both unfolding of the protein and its translocation are coupled to ATP
hydrolysis.
Even though the α and βsubunits are closely related in sequence, only the βsubunits
have protease activity. Chymotrypsin-like, trypsin-like protease activity, and an activity
that cleaves after glutamate residues have been identified. As the polypeptide is fed
through the 20S complex, it gets degraded by the βsubunits, and peptides of 7-10 amino
acids are released from the proteasome. Thus, the architecture of the proteasome
machine segregates the proteolytic component from the cytoplasm, and also prevents
cytosolic proteins from accidentally falling in and getting degraded.
An inhibitor of the proteasome has been identified. It is called lactacystin. Studies using
this inhibitor showed that most cytosolic proteins are degraded by the proteasome.
III. What about ER proteins?
So far we’ve seen that proteolysis in the cell takes place in the cytoplasm, albeit in
structurally confined spaces. Are there proteases in the ER that degrade misfolded
proteins? If so, how are they separated from resident and newly synthesized proteins?
Are there proteasomes in the ER? These large structures have never been observed in the
ER. Studies of human cytomegalovirus (HCMV) suggest that proteins that aren’t
properly folded, or not glycosylated correctly are secreted out of the ER by retrograde
transport.
When most viruses infect a cell, the cell responds by putting some of the foreign protein
on a flagpole on the surface of the cell to tell T cells to come kill it. These flagpoles are
known as major histocompatibility (MHC) antigens. MHC class I heavy chains bind to
peptides of about 7-9 amino acids (yes, you guessed it, produced by the proteasome) and
its partner β2-microglobulin. This complex is transported to the surface of the cell. In
HCMV infected cells, the heavy chains are co-translationally transported into the ER and
glycosylated, as in normal uninfected cells, but then are quickly deglycosylated,
transported back to the cytoplasm and degraded. This degradation is inhibited by
lactacystin, suggesting the proteasome is involved. Furthermore, researchers have been
able to detect an association between Sec61p and the heavy chain. Since proteasomes
have also been observed to be closely associated with ER membranes, one model is that
retrograde transport of errant polypeptides from the ER is tightly coupled to degradation
by the proteasome.
How the ER distinguishes a protein that is in the process of folding from those that are
not able to fold, and how these errant proteins are delivered to the translocation
machinery to be exported back to the cytoplasm is not known.
IV. The Unfolded Protein Response
What we do know is an interesting phenomenon known as the unfolded protein response
(UPR). When a lot of “junk” proteins accumulate in the cell, for example by treating
cells with reducing agents, or by preventing glycosylation, the ER sends out a signal that
upregulates resident ER proteins. These include BiP, protein disulfide isomerase, and
peptidyl-prolyl isomerase, which as you learnt last time, help proper folding of proteins.
The mechanism by which this response is mediated is very interesting.
All the genes that are upregulated in the UPR have a cis-acting element in their control
region that is bound by the transcription factor HAC1 (refer to the handout). During
normal growth, HAC1 is produced but quickly degraded by the ubiquitin/proteasome
pathway. However, during UPR, the mRNA of HAC1 is spliced and produces a stable
protein that can then upregulate the response genes. It turns out that an ER
transmembrane protein, Ire1p, somehow senses the abundance of junk in the ER. The
cytosolic side of this protein is partly a serine/threonine kinase, and partly an
endoribonuclease. Ire1p is responsible for cleaving the HAC1 mRNA, which is then
ligated by RLG1, a tRNA ligase.