Parkinson's and 3D structures

Cause of PD

Loss of dopamine producing neurons in substantia nigra/midbrain

Symptoms of PD

Tremor, difficulty with movement

Base treatment of PD

Dopamine precursor supplementation (L-DOPA)

Why L-DOPA and not dopamine?

Dopamine doesn’t cross the barrier

Does L-DOPA treat DP?

It only alleviates the symptoms

Clues to understanding molecular mechanisms

Pathogenicity and genetics

Pathogenicity

Hallmarks (pathways) of neurons that make them vulnerable

Genetics

1. Look into genetic mutations

2. Look into the functions of the mutated genes

Pathway through pathogenicity

PD and mitochondrial damage

Story behind that pathway

MPTP in MPPP (recreational drug)

What is MPTP?

Inhibitor of mitochondrial respiration/of one of the proteins in the chain (Complex 1)

Result of MPTP poisoning

Loss of SNc DA neurons bc of mitochondrial inhibition

Why did SNc DA neurons die?

Bc of their high metabolism (high dependency on mitochondria)

What other aspect of mitochondria affects neurons?

Mitochondrial respiration creates oxidative stress = damaged mtDNA and proteins

Pathway through genetics

Over 40 genes associated

Monogenic form of PD (5-20%)

Only one gene mutation is sufficient to cause the disease

Most important onset factor

Age

Early-onset cases

Under 50 yo

Late-onset

Over 60 yo

Are the targeted genes only expressed in neurons?

No: everywhere in the body

Which gene mutations cause early-onset recessive (both alleles) PD?

Parkin (120m) and PINK1 (200m)

Mutations in Parkin and PINK1 cause…

• 1% of all PD cases

• More than 60% of early-onset cases

How can mitochondria be damaged?

Mitochondrial depolarization by CCCP or respiration inhibitation by MPTP/rotedone

Result of damage

Recruitment of Parkin by mitochondria

Class of PINK1

Enzyme/Protein kinase that phosphorylates ubiquitin in damaged mitochondria

How is Parkin recruited to mitochondria?

P-ubiquitin recruits it and activates it

Activity of Parkin

Adds ubiquitin to mitochindrial proteins = damaged mitochondria

Approach to test if PINK1-Parkin mt quality control is defective

Structural biology for mechanistic insights

How do we get 3D atomic model of a protein?

X-ray crystallography

X-ray crystallography

Protein crystals that are frozen in a loop and exposed to high-intensity X-rays = electron density map from the diffraction pattern

Requirements of X-Ray crystallography

1. High content of purified proteins

2. Proteins conditions that yield crystals

Key element of crystals

The protein copies must all have the same orientation in the crystal

AI and 3D atomic models

Ai can predict it but not 100% accurate

Advantages of X-Ray crystallography (2)

1. Domain organization

2. Catalytic site localization (RING2-RING0 domains interface)

Where does the ubiquitin from Parkin come from?

Catalysed transfer from an E2 enzyme

Intermediate of the transfer

Thioester on RING2 (linked to the earliest case of PD (18 yo))

How is Parkin auto-inhibited?

1. Distance between E2-site (RING1) and catalytic site (RING2) is too long (50 Å)

2. REP blocks access to E2-site (gatekeeper)

Hypothesis to release auto-inhibition

Mutation of chain residue that anchors REP: Trp403 into Ala/W403A = accelerated recruitment

Role of Lys161/211

These residues are +ve, bind the phosphate and are mutated in PD = therefore needed for Parkin activation through phosphorylation

PD mutations that can be rescued by activating W403A mutation

Missense mutations: K161N and K221N

Activating mutations that rescue

W403A and F146A

PD mutations that cannot be rescued

Active site mutations (C431F) and E2-binding site mutations (T240R)