AMYLOID INDUCTION AND INHIBITION

Ayesha Zainab Beg14 MBT 007

CONTENTSIntroductionWhen protein loses its native form!AmyloidInductionInhibitionConclusion

IINTRODUCTION

Protein when loses its native conformation tends to retreat to aggregation.

One of the insoluble aggregate is amyloid, a β sheet rich fibrillar conformant.

The accumulation of amyloid in tissue gives rise to amyloidosis, which leads to toxicity.

Amyloid is induced by many factors which include predominantly misfolding and genetics .

Amyloid can be inhibited at many stages.

Native

Intermediate

Unfolded

Ordered aggregates

Amorphous aggregates

WHEN PROTEIN LOSES ITS NATIVE FORM!

Overview of protein folding machinery

Quality control escape

Amorphous and fibrillar aggregates

Energy landscape scheme of protein folding and aggregation.

AMYLOIDS

conformation.Virchow first identified substance having affinity for iodine as that of starchCongo red dye gives green birefringence under cross polarized light.

Electron microscopy confirmed fibrillar form of amyloid.

X ray diffraction showed that fibrils were composed of polypeptide chain in the β cross form.

AMYLOIDSInsoluble protein aggregates 80-100 A thick fibrils.

Amyloids contd..

Amyloid fibrils are about 7- 10 nm in their diameter and are composed of 2-6 protofilament.

The β strands are oriented perpendicular to, and the β sheets parallel to the fibril axis.

The two sheets intercalate to form zipper which leads to the formation of amyloid fibrils.

Amyloid fibrils show a high degree of uniformity.

Mechanism of Amyloid Generation

INDUCTION

Amyloid

Protein misfolding

Genetic mutationOthers

PROTEIN MISFOLDING DEPENDENT INDUCTION

Over expression of protein leading to aggregation

Inefficient Chaperones

Inefficient enzymes PDI

Impaired Ubiquitin-Protesome System

• Protein over-expression increases the concentration of polypeptide chains driving the polypeptide chain into an aggregation pathway as well.

• It is noteworthy that folding of a monomeric protein is a zero-order reaction whereas aggregation is at least a first-order reaction.This means that increasing protein concentration may force a protein into an aggregation route..

MONOMERS

OVER EXPRESSED PROTEIN

AGGREGATION

Quintas A. What drives an amyloid protein precursor from an amyloidogenic to a native-like aggregation pathway? OA Biochemistry 2013 Mar 01;1(1):6.

OVEREXPRESSION OF PROTEINS

• Mutated or inefficient chaperones Hsp70/40 and Hsp 90 lead to early stages of amyloid formation.

• Hsp70 blocks aggregation of α-synuclein, in Parkinson disease, in vitro .

• Addition of Hsp70 and Hsp40, to Huntingtin, an aggregation prone protein that causes Huntington disease, blocks oligomer formation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 44, pp. 33182–33191, November 3, 2006

INEFFICIENT CHAPERONES

• Sequestration of proteasomes into aggregate.

• Proteasomes unable to cleave within polyQ sequences, and proteasomes may become clogged by these fragments or release polyQ peptides when polyQ-expanded proteins are inefficiently degraded. 

J o u r n al o f C ell S cie n c e 1 2 2 , 3 2 6 2 - 3 2 7 1 P u blis h e d by T h e C o m p a ny o f Biolo gis t s 2 0 0 9

IMPAIRED UBIQUITIN-PROTESOME SYSTEM

Front. Mol. Neurosci., 29 September 2014

NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of aggregated proteins,

Volume 586, Issue 18, 31 August 2012, Pages 2826–2834 FEBS LETTERSNature Neuroscience 9, 865 - 867 (2006) 

INEFFICIENT ENZYMES PDI

GENETIC DEPENDENT INDUCTION

Three types of genetic abnormalities have been identified in amyloidogenic proteins:

Variant molecules ( due to missense mutations, deletions, or premature stop codons)(AD and FBD)

Genetically determined posttranslational modifications (FAF- Gelsolin)

Mutations in genes for non-amyloidogenic proteins can play a permissive role in amyloid development(TTR-FAP V30M). 

•Early onset AD (EOAD)

•Mendelian Autosomal Dominant Inheritance

•Linkage studies identified 3 genes for multiple mutation ( APP, PSEN1, PSEN2).

•Increased Aβ40 and Aβ42 , aggregation and increased β and γ secretase

MUTATION

Non-amyloidogenic protein point mutation(TTR)

Genetically determined posttranlational modification

V30M POINT MUTATION

ENVIRONMENTAL AND OTHER FACTORS Deamidation of glutamine and

asparagine ,thermodynamic stability, native folding and flexibility of molecule is changed ,net charge on the protein is changed closer to zero leading to protein aggregation.

Oxidative damage- (LOAD), lipid peroxide act with Cys, His, Lys residues generating advanced lipoxidation end-products, change hydrophobicity and crosslinking.

Change in net charge due to pH change.

INHIBITION

INHIBITORY MOLECULES

Small compounds that

inhibit aggregation;

Immunotherapy

Compounds that interfere with amyloid-

cell or amyloid-protein

interactions

Pharmacological and chemical

chaperones

INHIBITION LEVELS

RNAi ( post-

transcriptional level)

Stablizing native state (kinetic stablizers of

native state ttr tafamidis )

Inhibition of aggregation and

fibrils

Amyloid fibrils and misfolded

protein

mRNA

RNAi therapeutic drug administration

STABLIZING NATIVE STATE

Native structure stabilized by small compounds

•Christine E. Bulawa,  9629–9634, doi: 10.1073/pnas.1121005109•Christine E. Bulawa,  9629–9634, doi: 10.1073/pnas.1121005109•Christine E. Bulawa,  9629–9634, doi: 10.1073/pnas.1121005109

Christine E. Bulawa,  9629–9634, doi: 10.1073/pnas.1121005109

INHIBITION OF AGGREGATION AND FIBRILS

Small molecules inhibited the oligomerization of monomers and disrupted the firillogenesis(others- Gallic derived product)

NATURE Scientific Reports Article number:7992 doi:10.1038/srep07992 Published 23 January 2015

AMYLOID FIBRILS AND MISFOLDED PROTEIN

Immunotherapy

Targets amyloid fibrils and also clears off-pathway aggregates.

Inhibition of Amyloid Formation J. Mol. Biol. (2012) 421, 441–465

CONCLUSION

Amyloids are complex aggregates induced by many factors leading to systemic and localised amyloidosis.

Amyloids are extensive source of neurodegenerative disorders related to more than 10 diseases.

Amyloid inhibition is extensively researched at different levels, leading to the discovery of edible blockers.

Pharmacological chaperones, immunotherapeutics , small molecules and RNAi mediated inhibition is being extensively studied and are showing promising results.

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