BINF

1
Protein Function PROTEIN DEGRADATION
Lecture 14 BINF 5513
2
Explain this figure.
3

Explain this figure.
4
Traditional single molecule way to integrate
evidence describe function
EF2_YEAST
Descriptive Name Elongation Factor 2
Lots of references to papers
Summary sentence describing functionThis
protein promotes the GTP-dependent translocation
of the nascent protein chain from the A-site to
the P-site of the ribosome.
5
(No Transcript)
6
In the past few decades biochemistry has come a
long way towards explaining how the cell produces
all its various proteins.
But as to the breaking down of proteins Today we
will discuss one of the cell's most important
cyclical processes, regulated protein degradation
7
Some properties of intracellular protein
degradation. Abnormal proteins are rapidly
eliminated. Normal proteins are selectively
degraded at widely different rates. Levels of
specific proteins in animal cells can be
regulated by changes in rates of synthesis or
rates of degradation. Proteins are degraded
into amino acids.
8

• PROTEIN DEGRADATION1
• Biochemical degradation of protein through
  hydrolysis of peptide bonds

H
C
N
O
9
Turnover of proteins Synthesis gt Active Protein
gt Degradation Amino acids used for
synthesizing proteins are obtained by degrading
other proteins. Glucose, fatty acids and ketone
bodies can be formed from amino acids. Excess
amino acids cannot be stored. Surplus amino
acids are used for fuel.
10
Protein Turnover In a typical day, a person will

consume 100 grams of protein, break
down 400 grams of bodily protein,
resynthesize 400
grams of protein,
excrete/catabolize 100
grams.
11

• Protein Degradation
• The functions of intracellular proteolysis-degrada
  tion are
• the elimination of abnormal proteins,
• the maintenance of amino acid pools in cells
• selective destruction of proteins whose
  concentrations must
• vary with time and alterations in the state of a
  cell.

Thus cellular proteins are source of amino
acids Dietary proteins are another vital source
of amino acids
12
Dietary Protein Degradation - Serine Proteases A
number of simple protein-degrading enzymes were
known trypsin, Chymotrypsin, and Elastase. They
break down proteins in our food to amino acids in
the small intestine and amino acids are absorbed
into the bloodstream
Intestine - part of the canal between the stomach
and the anus. ( Digestive apparatus)
Enzymes aa sequences are very similar with 62 aa
the same out of 257. Three key amino acids
(His-57, Asp-102 and Ser-195) involved in the
mechanism of catalasis
His-57 Asp-102 Ser-195
13
Site-directed mutagenesis can identify residues
which involved in the Catalytic Mechanism
It was shown Asn102 Mutant of Trypsin lost most
of its activity.
These results show that Asp102 is essential for
trypsin catalytic activity.
14
Chymotrypsin vs Trypsin

• The asterisks mark identical residues, and the
  dots mark very similar side chains, such as Leu
  and Ile,

We see from the alignment that the proteases are
homologous.
15
The 3-D structures are very similar - little
alpha-helix - and a central beta sheet.
All three enzymes hydrolyze the peptide bond)
but they have different target aas.
Trypsin ? Arg, Lys Chymotrypsin ? Phe, Tyr,
Trp Elastase ? Ala, Ser, Val, Thr
Structural superposition
To accommodate these different substrates, the
substrate binding pocket is adjusted in each
enzyme.
Trypsin The
positively charged Lys and Arg of the target
amino acid fits into a deep pocket with a
negatively charged amino acid (Asp-189).
16
When a substrate binds to the enzyme, it "fits"
into the active site.
The enzyme-substrate complex then enters
into a transition state where the substrate's
bonds are more easily broken (lowered activation
energy). Once the reaction occurs, the active
site is altered, releasing the product.
Each enzyme works at precise pH, temperature and
chemical conditions, such as the amount of sodium
ions in the cell.
17
Another example of protein degradation is in the
lysosome - a small sac-like membrane bound
compartment inside a cell in which proteins
absorbed from outside are broken down. lysosome
contain over 50 different digestive (hydrolytic
enzymes). The internal pH of lysosomes is low
(pH 5). This is optimal for activity of
hydrolytic enzymes. The low pH optimum of
hydrolytic enzymes protects the components of the
cytoplasm (pH 7.2) from being degraded

Common to the processes of degradation in the
Intestine and lysosome that they do not require
energy.
18

19
In contrast, other cellular protein degradation
processes does require energy.

Cellular Protein Degradation

• Cellular proteins are degraded at different
  rates.
• Ornithine decarboxylase has a half-life of 11
  minutes.
• Hemoglobin lasts as long as a red blood cell.
• ?-Crystallin (eye lens protein) lasts as long as
  the organism does.

20
Question What conditions do long-lived and
short-lived proteins depend on?
Answer Conditionally unstable proteins,
long-lived or short-lived proteins depending on
the state of a cell.
The condition of degradation are often
deployed as components of cell control. Example
cyclinsa family of proteins whose destruction at
specific stages of the cell cycle regulates cell
division and growth. Another Example many
proteins are long-lived as components of larger
complexes such as ribosomes but are unstable as
free subunits.
21
Cellular Protein Degradation is a multi-step
reactions process. The first step is a labeling
a protein, which should be destroy. This reaction
enables the cell to break down unwanted proteins
with high specificity.
The labeling requires energy. The label is
ubiquitin -

76-amino-acid-long polypeptide The molecule was
found in numerous different tissues and organisms
it was given the name ubiquitin
(from Latin ubique,
"everywhere")
Ubiquitin - a polypeptide that represents the
"kiss of death".
22
How does Ubiquitin label a protein? Features of
proteins that confer instability are called
degradation signals, or degrons. The essential
component of one degradation signal, the first to
be discovered, is a destabilizing N-terminal
residue of a protein. This signal is called the
N-degron.
A. VARSHAVSKY, Proc. Natl. Acad. Sci. USA, 1996
The N-End Rule. A relation between the metabolic
stability of a protein and the identity of its
N-terminal residue.
23
Approximate in vivo half-lives of ?-galactosidase
proteins in
E. coli and in
S. cerevisiae Arg, Lys, Phe, Leu, Trp, TyrThe
presence of one of these amino acids at the
N-terminus gives a protein a half life of 2-3
minutes in bacteria or eukaryotes
24
Ubiquitin can form a very stable chemical bond
with various proteins. This is the triggering
signal that leads to degradation of the protein.
It is this reaction that constitutes the actual
labeling, the "kiss of death "the multistep
ubiquitin-tagging hypothesis" based on three
enzyme termed E1, E2 and E3.
Destruction of a targetted polypeptide involves
Recognition by a three enzyme system which
attaches the protein ubiquitin to the target.
The signal is reinforced by adding additional
copies of ubiquitin to form a polyubiquitin tag.
Polyubiquitin tagged protein is delivered to a
structure called the proteasome, which breaks
down the polypeptide into short oligopeptides.
25
Ubiquitin-mediated protein degradation
1
2
1) Ubiquitin is activated at its C-terminal by
the ubiquitin activating enzyme, E1.
(This reaction requires energy in the form
of ATP) Ub-CO2- ATP E1 ? Ub-CO-E1 A
cell contains one or a few different E1 enzymes
3
4
5
6
26
2) Ubiquitin is then transferred to ubiquitin
conjugating protein E2. (E1 and E2 bound
covalently to the ubiquitin.
Binding required ATP
Ub-CO-E1 E2 ? Ub-E2
E1 A cell contains some tens of E2 enzymes
1
2
3
4
5
6
27
3) The E3 enzyme can recognize the protein target
which is to be destroyed. It is the specificity
of the E3 enzyme that determines which proteins
in the cell are to be marked for destruction in
the proteasomes. E3 binds to the primary
N-terminal degradation signal of target
polypeptides. E3 catalyses the transfer of
labeled ubiquitin from E2 to the protein
substrate.
1
2
3
4
5
6
28
4) Polyubiquitination. the additional Ub units
being added to the previous Ub to form an
oligo-Ub chain 5) This ubiquitin chain is
recognised in the opening of the proteasome and
the ubiquitin label is
disconnected and the protein is admitted. 6)
What is a proteasome?
1
2
3
4
5
cells break down up to 30 of the
newly-synthesised proteins via the proteasomes
since they do not pass the cell's rigorous
quality control.
6
29
The proteasome the cell's waste
disposer Proteasomes are barrel-formed
structures. The black spots indicate active,
protein-degrading surfaces within the barrel
where it is shielded from the rest of the cell.
The only way in to the active surface is via the
"lock", which recognises polyubiquitinated
proteins.
A human cell contains about 30,000 proteasomes
The proteosomes can break down practically all
proteins to 7-9-amino-acid-long peptides. The
peptides are released from the other end of the
proteasome.
The proteasome itself cannot choose proteins,
only ubiquitin-labelling proteins.
30
6) Proteasome-degradation.
In eukaryotes 20S proteasome is able to break
down unfolded target polypeptide. In eukaryotes
26S proteasome is required to attack a folded
protein.
Groll, M. et al. (1999) The catalytic sites of
20S proteasomes and their role in subunit
maturation. A mutational and crystallographic
study. PNAS 96 10976
31
Now we can more understand the role of
ubiquitin-labelled protein degradation. For
example, a mutated cell is needed in the
ubiquitin system. Why? It was shown that
ubiquitin-controlled protein breakdown was not
only important for degrading incorrect proteins
in the cell but it also take part in control of
the cell cycle, DNA replication, chromosome
structure.
32
The ubiquitin system is involved in the
regulation of
the cell cycle The E3 enzyme "anaphase-promoting
complex" (APC) plays an important role in the
separation of the chromosomes during mitosis and
meiosis.
Anaphase - pull the
chromosomes away - stage
33
Once the rope is gone, the
chromosome pair can be separated.
A protein complex acts like a rope around the
chromosome pair, holding it together.
The inhibitor doesnt allow to destroy a rope
At a given signal, the APC labels an inhibitor
The enzyme is released, is activated and cuts the
rope around the chromosome pair
and
the inhibitor is carried to the proteasome and
destroyed.
As the result
34
Incorrect chromosome division during meiosis is
the commonest cause of spontaneous miscarriage
during pregnancy -
an
extra chromosome 21 in humans leads to Down's
syndrome. Most malignant tumours have cells with
changed numbers of chromosomes as a result of
incorrect chromosome division during mitosis.
35
The ubiquitin system is involved cancer and
programmed cell death E3 enzyme and the p53
protein p53 - a tumor suppressor protein that
normally inhibits the growth of tumors. Damages
in the p53 protein are considered as cause for
50 of all human cancers. Like other
tumor-suppressor genes, p53 normally controls
cell growth. If p53 is physically lost or is not
functioning (because it has been inactivated),
this may permit the cell to divide without
restraint. the p53 protein has been called the
guardian of the
genome
36
In health p53 is continually produced and
degraded in the cell. The breakdown is regulated
through ubiquitination and the E3 enzyme forms a
complex with protein p53.
Following DNA injury, protein p53 is
phosphorylated and can no longer bind to its E3
enzyme.
The breakdown stops and
the quantity of p53 in the cell rises rapidly.
37
Normal Protein p53 acts as a transcription
factor, i.e. a protein that controls the
expression of certain genes that regulate DNA
repair and programmed cell death. Raised levels
of protein p53 lead first to interruption of the
cell cycle to allow time for repair of DNA
damage. However, If the damage is too extensive
the cell triggers programmed cell death and
"commits suicide".
38
p53 interacting with DNA is central to the normal
functioning of p53 as a tumor suppressor

• The position of the six hot spot amino acid
  residues (yellow). Mutations in hot spot amino
  acids either interfere with protein-DNA contacts,
  or disrupt integrity of the domain.