Protein aggregation is the most discussed topic as it is being linked to many neurodegenerative diseases. Here, in these slides I have tried to explain about protein aggregation and its mechanism.

1. MD NADIR HASSAN

2. CONTENT
 Protein???
 Misfolding and aggregation
 Are all amino acids equally prone?
 Mechanism of aggregation
 Fate of protein
 Protein Quality Control System
 Causes
 Factors affecting aggregation
 Evolutionary strategies
 Techniques used for characterisation
 Therapeutic strategies
 Conclusion

3. PROTEIN???
• The basic building block of proteins and change in the
linear sequence of amino acids leads to the formation of a
variety of proteins.
• There are thousands of protein present inside the cell in
living systems with each projecting a unique function.
• From having a role in DNA replication to a role in
catalysing the metabolic reactions or to act as transporting
molecules or responding to different stimuli, cellular
proteins perform numerous functions.

4. The function of protein is dependent on the structure of the
protein. There are four levels of protein structure

5. MISFOLDING & AGGREGATION
• Misfolding can be defined in a level that has an enormous
amount of non-native interactions between residues and their
properties differ from those of a similar state having highly
native-like interactions.
• The ‘Protein aggregation’ is defined as summary of protein
species of higher molecular weight such as ‘oligomers’ or
‘multimers’ instead of desired defined species( eg a monomer).
• Protein aggregation is presently considered a pathway
alternative to protein folding where intermolecular, rather than
intramolecular interactions are favoured.

6. CONTINUED.....
• The molecular basis of protein aggregation is protein mis-
folding, where a specific polypeptide chain loses, or is unable
to attain its native, closely packed 3D structure, thus
populating unfolded, partially folded or non correctly folded
states in equilibrium to each other.
• In these native states, hydrophobic core become exposed to the
solvent and it enhances the tendency to nucleate initial
oligomeric assemblies.

8. ARE ALLAMINO ACIDS EQUALLY
PRONE?
• Any protein can aggregate under suitable conditions, but the
propensity is modulated by the sequence of amino acids
• Certain regions in the protein known as ‘hotspots’ are more
prone to form aggregates.
• Hotspots are rich in hydrophobic and aliphatic aminoacid
residues.
• Hydrophobic residues with high propensities for betastrand
conformation and residues with complementary charges
promote fibril formation(**)
** Lars Tjernberg,Waltteri Hosia, Niklas Bark3,Johan Thyberg and Jan Johansson,
Charge attraction and beta propensity are necessary for amyloid fibril formation
from tetrapeptides JBC Papers in Press. Published on September 4, 2002

9. CONTINUED...
• The molecular basis of protein aggregation is protein
misfolding , where a specific polypeptide chain loses,or is
unable to attain its native, closely packed 3D structure,thus
populating unfolded,partially folded or non correctly folded
states in equilibrium to each other.
• In these native states,hydrophobic core become exposed to the
solvent and it enhances the tendency to nucleate initial
oligomeric assemblies.

10. MECHANISM OF AGGREGATION
• There is not a single defined mechanism for the protein
aggregation. There is interdependency among the mechanism
leading to several process/steps to be in common.
• There is a possibility of the same mechanism for different proteins
or one protein may undergo different mechanism(29). Some of the
mechanisms are:
• 1. Self-assembly of monomeric protein:
 Small reversible monomers are formed due to the self-
complementary nature and intermolecular interactions of the
surface of the monomer. And with an increase in protein
concentrations, large oligomers are formed

11. CONTINUED....
 In 1952, it was proposed that unfolding of proteins is not a
prerequisite condition to form amyloid aggregates; instead, the
side-by-side or end-to-end union of protein molecules results
in the aggregation of globular proteins.
• 2. Aggregation of conformationally altered monomeric
protein:
 Sometimes protein with altered conformation or in the
partially unfolded state has strong propensity to form higher
order oligomers rather than the self-association of the native
protein. So, there will be a transition from native to a non-
native structure which makes it different from the above.

12.  Stress in the form of heat or shear plays a vital role in this
mechanism. Interferon-ϒ (32) and Granulocyte-colony
stimulating factor (G-CSF) (33) have been reported to favour
this mechanism.

13. CONTINUED....
• 3. Nucleation and seeding mechanism
 In this mechanism, the native monomer alone cannot seed
the phenomenon of fibrillation, but aggregates of certain
critical size promote the formation of aggregates of
progressively larger size by adding monomers. This is termed
as a ‘critical nucleus’.
 The nucleation mechanism usually exhibits a lag phase
without any visible precipitate for a long period of time, but
after this critical period, a much larger species appears
instantly. This process of aggregation is termed as
‘homogeneous nucleus’ wherein the critical nucleus is itself
the product aggregate.
 In the ‘heterogeneous nucleation’, moieties other than
protein aggregates form a critical nucleus.
 Generally, amyloid formation occurs via nucleation-
dependent oligomerisation.

14.  After the establishment of the nucleus, the fibril growth occurs
very rapidly.
 The time gap between the formation of monomer and the
nucleus is known as the lag phase.

15. • 3D domain swapping
mechanism
 Identical protein replace
their domain.
 An interwined dimer or
higher order oligomer,
with one domain of each
subunit replaced by the
identical domain of other
subunit.
 Swapped domain may be
α-helix or β-sheet or an
entire tertiary globular
domain.

16. S.NO PROPOSED
MECHANISM
PROTEINS YEAR
1 End to end or Side by
side addition of
monomer
Albumins 1953
2 Reversible growth
mechanism
Glutamate
dehydrogenase
1970
3 Nucleation
mechanism
Actin 1975
4 Prion aggregation
mechanism
Prion 1991
5 Two steps model of
nucleation
Amyloid beta 1997
6 Association of
conformationally
altered monomer
Amyloid beta 2004
7 Secondary
nucleation
mechanism
Amyloid beta 2013
SOURCE-** M.K.Siddiqi, P.Alam, S.K.Chaturvedi, Y.E.Shahein, R.H.Khan 2017
Mechanisms of protein aggregation and inhibition. Frontiers in Bioscience, Elite, 9, 1-20

17. FATE OF PROTEIN

18. PROTEIN QUALITY CONTROL SYSTEM

19. CAUSES OF PROTEIN AGGREGATION
• Protein aggregation can occur due to a variety of causes.Some
of them are:
1. MUTATIONS
- When mutation in the DNA affect the sequence of amino
acids,a different amino acid can change the interaction
between the side chains that affect the folding of protein.
- Hydrophobic amino acids might get exposed and aggregation
may occur.
- Point mutations in causative proteins such which may leads
to
* Loss of cellular protein quality control system
*Inability of the ubiquitin proteasome complex to
degrade and eliminate misfolded aggregation-prone
molecules .

20. CONTINUED....
*Inefficient functioning of the molecular chaperone
machinery.
2. PROBLEM WITH PROTEIN MACHINERY
3. ENVIRONMENTAL SRESSES
* It includes high temperature and PH, oxidative stresses
which can also lead to protein aggregation.
* Extreme temperatures can weaken and destabilises the non
covalent interactions between amino acid residues.
* Oxidative stress can be caused by reactive oxygen
species.These unstable radicals can attack the amino acids
,leading to oxidation of side chains or cleavage of polypeptide
bonds.
4. AGING

21. FACTORS AFFECTING AGGREGATION

22. EVOLUTIONARY STRATEGIES
• Integral burial of Hydrophobic stretches in native protein.
• Protection by ‘gate-keeper’ residues such as glycine and
proline
• Alternating polar and nonpolar amino acids favour amyloid
formation and interestingly, sequences with such binary
patterns are seen to be rare in the database of natural proteins
• The free-edge strands of proteins are protected from non-
native intermolecular b-sheet interactions by the strategic
placement of prolines, charged residues

23. TECHNIQUES USED FOR
CHARCTERISATION

24. AMYLOID RELATED DISEASES

25. THERAPEUTIC STRATEGIES
The need of the hour is to have a therapy against protein
misfolding diseases, as protein misfolding and aggregation are
crucial in the pathogenesis of neurodegenerative diseases.
• Approaches can be
categorised into:
 Stabilization of native
protein conformation
 Blocking the aggregation
process
 Diminishing of aggregation-
prone species
 Increased clearance of the
misfolded protein

26. CONCLUSION
• Protein aggregation results in the formation of
amyloid fibrils and inclusion bodies and these are
associated with many diseases like neurodegenerative
disorder and prions related disease.
• It is now more evident that protein aggregates formed
in cells are also due to malfunctioning of protein
quality control systems like proteasome machinery
and autophagy.
• Apprehension of kinetics of amyloid formation and
pathways may help in designing strategies and
approaches that either lead to inhibition or reverse the
process of aggregate formation.

27. Thank you