description of protein degradation pathways in lysosomes and ubiquitin mediated pathway and n-end rule in different organisms

1. PROTEIN DEGRADATION
&
N-end rule
D.INDRAJA

2. • The levels of proteins within cells are determined not only by rates of
synthesis, but also by rates of degradation.
• The half-lives of proteins within cells vary widely, from minutes to several
days, and differential rates of protein degradation are an important aspect of
cell regulation.
• Many rapidly degraded proteins function as regulatory molecules, such
as transcription factors.
• Other proteins are rapidly degraded in response to specific signals, providing
another mechanism for the regulation of intracellular enzyme activity.
• I n addition, faulty or damaged proteins are recognized and rapidly
degraded within cells, thereby eliminating the consequences of mistakes
made during protein synthesis.
• In eukaryotic cells, two major pathways:
– lysosomal proteolysis—mediate protein degradation.
– ubiquitin-proteasome pathway

3. WHY?
• Cells also degrade other types of proteins while they are still
functional(remodeling involves proteolysis of one set of structural and
metabolic proteins and its replacement by another specialized for a different
purpose.)
• Removal of damaged or harmful proteins(proteins can be misfolded or
denature they may fail to assemble properly in to complexes,or they can be
altered by some abnormal post translational modifications. Such aberrant
proteins are potentially toxic and need to be eliminated)
• The eukaryotic cell has a remarkable ability to distinguish normal from
abnormal proteins and selectively degrade the letter
• When the capacity is compromised, disease often results

4. • The first type of protein degradation identified in the cell is lysosome and
autophagosome mediated degradation
Lysosomal proteolysis
• The lysosome is a membrane-bound intracellular compartment full of nonspecific
proteases that will cleave into individual amino acids any protein they come into
contact with. Proton pumps fill the lysosome with H+ from the cytosol, making it
acidic (pH 4.8) — the proteases function optimally at this pH and not at all at
cytosolic pH (7.2), thus minimizing the risk to the cell in the event of lysosome
rupture.
• The lysosome is formed by budding off from a compartment of the late Golgi – it
represents an alternate endpoint for some proteins in the secretary pathway that
neither stay in the ER or Golgi nor undergo exocytosis to the cell surface

5. • Proteins destined for lysosomal degradation can reach the lysosome by a
variety of means.
1. receptor-mediated endocytosis  endocytic vesicles from the cell surface
can fuse with the lysosome; this is a mechanism for degradation of cell
surface receptors and thus the down regulation of incoming signals
2. Phagocytosis the cell engulfs foreign bodies – say, invading bacteria, or
apoptotic bodies from other cells – and delivers them to the lysosome. The
membrane of the lysosome itself can invaginate, creating exosome-like
vesicles full of cytosolic proteins to be degraded
receptor-mediated endocytosis Phagocytosis

6. 3. “canonical”, starvation-induced autophagy, In this double membrane
forms around material (such as unneeded organelles) in the cytosol and
delivers them to the lysosome.
• The degradation of proteins in the lysosomes is catabolic – it releases energy
– so this response to nutrient starvation recovers some of the energy
originally put into synthesizing proteins and other cellular components.
• But autophagy isn’t induced only by starvation – unfolded protein stress in
the endoplasmic reticulum can cause chunks of ER to be degraded by
autophagy

7. • The proteins degraded by lysosomal proteases under the starvationconditions
contain amino acid sequences similar to the broad consensus sequence Lys-
Phe-Glu-Arg-Gln, which presumably targets them to lysosomes.
• A member of the Hsp70 family of molecular chaperones is also required for
the lysosomal degradation of these proteins, presumably acting to unfold
the polypeptide chains during their transport across the lysosomal
membrane.
• The proteins susceptible to degradation by this pathway are thought to be
normally long-lived but dispensable proteins.
• Under starvation conditions, these proteins are sacrificed to provide amino
acids and energy, allowing some basic metabolic processes to continue.

8. ubiquitin-proteasome pathway
• The major pathway of selective protein degradation in eukaryotic
cells uses ubiquitin as a marker that targets cytosolic and nuclear proteins for
rapid proteolysis
• Ubiquitin is a 76-amino-acid polypeptide that is highly conserved in all
eukaryotes (yeasts, animals, and plants). Proteins are marked for degradation
by linkage occurs between the carboxyl group of the C-terminal glycine
residue of ubiquitin and the E aminogroup of a lysine residue within the
substrate forming a type of amide bond often referred to as an isopeptide
bond.

9. POLYUBIQUITINATION
• Additional ubiquitins are then added to form a multi ubiquitin chain.
• Such polyubiquinated proteins are recognized and degraded by a large,
multisubunit protease complex, called the proteasome.
• Ubiquitin is released in the process, so it can be reused in another cycle. It is
noteworthy that both the attachment of ubiquitin and the degradation of
marked proteins require energy in the form of ATP.

10. • Ubiquitination is a multistep process. First, ubiquitin is activated by being
attached to the ubiquitin-activating enzyme, E1. The ubiquitin is then
transferred to a second enzyme, called ubiquitin-conjugating enzyme (E2).
The final transfer of ubiquitin to the target protein is then mediated by a
third enzyme, called ubiquitin ligase or E3, which is responsible for the
selective recognition of appropriate substrate proteins.

11. • In some cases, the ubiquitin is
first transferred from E2 to E3
and then to the target protein .
In other cases, the ubiquitin
may be transferred directly
from E2 to the target protein
in a complex with E3.
• Proteins in the other major
class of E3 have a single
subunit with a segment called
the HECT domain. This
domain act as a direct carriers
of the activated ubiquitin
• For the HECT E3s the
activated ubiquitin is
transferred from the E2to a
conserved cysteine side chain
in the HECT domain

12. Structure of E3
• The E3 protein contains a structural motif called a RING FINGER that constitutes
either a motif with in a larger polypeptide or one of several subunits within a
complex
• Most cells contain a single E1, but have many E2s and multiple families of
E3 enzymes. (E1<E2<E3)

13. • It has different substrate binding subunits can be used with the same
framework subunit producing E3s with similar architecture but different
substrate specificities
• The variable subunits not only provide sites for binding different substrates
but also produce gaps of different sizes between the site and the E2.
• This presumably helps the E3 adapt to substrates of different dimensions

14. Recognition of degron by
E3
• A PEST sequence is a peptide
sequence that is rich
in proline (P), glutamic
acid (E), serine (S),
and threonine (T). This sequence is
associated with proteins that have a
short intracellular half-life;
therefore, it is hypothesized that
the PEST sequence acts as a signal
peptide for protein degradation.
• This protein degradation may be
mediated via the proteasome
• The degrons may be degraded by
specific signals such as
phosphorylation, methylation,
glycosylation exposure of
hydrophobic molecules outside of
the protein
• Phosphorylation is the most
common degron modification that
triggers substrate binding to E3

15. Hidden degrons
• Some times the degrons may be hidden due to complex formation so that
they live in the cell at a longer time than the ususal
• Eg: phosphorylation dependent degron found in a yeast inhibitor of the onset
of DNA replication. Atleast six of nine potential sites in this inhibitor must
be phosphorylated by a cyclin dependent kinase before the inhibitor is
recognized by an E3 ligase and ubiquinated

16. proteasome
• The proteasome is a cylindrical protein complex found in the cytosol which
cleaves up proteins tagged with ubiquitin.
• The proteasome itself weighs in at 26S, and is composed of a 19S gate and a
20S core. The 19S gate recognizes and binds ubiquitinated proteins,
powered by ATP – unlike the lysosome, the proteasome is an energy-losing
operation
• The 19S subunits act as a gates for entry and exit site of proteins. which has
lid and a base
• Lid deubiquination
• Baseunfolding

17. • Once recognized, these proteins must be de-ubiquitinated and unfold in
order to pass through the narrow channel of the 19S and enter the 20S core,
a cylindrical complex which does the actual chopping up of proteins.
• Unlike the lysosome, where proteases shear proteins up into individual
amino acids, the proteasome just chops proteins into small peptides, usually
of 7 – 9 amino acids each.

18. Active site of proteolysis
• Central part which is a processive proteolysis part has a two α and two β
types they are seven subunits in both α and β subunits
• This central cylinder has a certain pH and temp that is required to degrade a
protein
• They are three types of active sites in the β subunits, each with a different
specificity, but all employ an N-terminal threonine.
• The hydroxyl group of the threonine residue is converted in to a nucleophile
that attacks the carbonyl groups of peptide bonds to form acyl-enzyme
intermediates

19. Path of protein degradation
•In the path of protein degradtaion the ATP is required for the entry of protein
only

20. ubiquitin-proteasome pathway

21. N-END RULE
• The N-end rule is a rule that governs the rate of protein degradation through
recognition of the N-terminal residue of proteins.
• The rule states that the N-terminal amino acid of a protein determines its
half-life (time after which half of the total amount of a given polypeptide is
degraded).
• The rule applies to both eukaryotic and prokaryotic organisms, but with
different strength, rules, and outcome.
• In eukaryotic cells, these N-terminal residues are recognized and targeted
by ubiquitin ligases, mediating ubiquitination thereby marking the protein
for degradation
Discovery
• The rule was initially discovered by Alexander Varshavsky and co-workers
in 1986.

22. • Rules in different organisms
Aminoacids present Half-life
Met, Gly, Ala, Ser, Thr, Val, Pro > 20 hrs (stabilizing)
Ile, Glu approx. 30 min (stabilizing)
Tyr, Gln approx. 10 min (destabilizing
Leu, Phe, Asp, Lys approx. 3 min (destabilizing)
Arg approx. 2 min (destabilizing)
For s.cervisiae
For Mammals

23. Bacteria
• In Escherichia coli, positively-charged and some aliphatic and aromatic
residues on the N-terminus, such as arginine, lysine, leucine, phenylalanine,
tyrosine, and tryptophan, have short half-lives of around 2-minutes and are
rapidly degraded.
• Other amino acids on the other hand may have half-lives of more than 10
hours when added to the N-terminal of the same protein
• However, a complicating issue is that the first residue of bacterial proteins is
normally expressed with an N-terminal formylmethionine (f-Met).
• Once the f-Met is removed, the second residue becomes the N-terminal
residue and are subject to the N-end rule.