FMD Journal Club Prep-session

Basic Science Journal Club 2

November 3rd, 2023

Objectives

2

• encourage critical thinking and understanding the basic science
• see the potential and pitfalls of translational research in the future
• today: Cystic Fibrosis (CF) – Targeting Protein Misfolding to Treat Disease

​

• Basic concepts of cellular trafficking including endoplasmic reticulum (ER) and Golgi complex
• Handing of misfolded proteins
• Appreciate the series of studies for clinical therapy of CF

What is cystic fibrosis

• genetic disorder, fatal in childhood
• Clinical manifestations: Malabsorption, recurrent lung infections, decrease in fertility, diabetes
• hallmark: “sweat chloride” condition – high levels of chloride ions in sweat
• This causes dehydration of epithelia resulting in abnormal mucus
• Until the cause was known, it was mostly symptomatic treatment – this would prolong the patient’s life expectancy
• Cause of death: respiratory failure

3

Discovery: CFTR gene

• Cystic Fibrosis Transmembrane Conductance Regulator gene
• encodes a protein that plays a crucial role in regulating the movement of chloride ions across cell membranes
• 1989 by Lap-Chee Tsui at UofT
• In parallel with Francis Collins in University of Michigan
• At that time life expectancy was 12 years in the US, and around 20 in Canada
• Now is typically ~50 with some patients living into their 80s

4

Dr. Lap-Chee Tsui at work in lab, 1988 – Image Credit The Hospital For Sick Children

Discovery: CFTR gene mutations

5

• Now we can study this gene - express the protein and make antibodies – we can find the protein and see what happens to in different mutations

• Defect Class I: make no protein at all
• Class II - most common for the CF phenotype: protein made in ER but never leaves ER (is degraded)
• Class III: protein not functional, IV-V: suboptimal function of the CF protein even if it is on the cell surface
• Class VI: more rapid degradation, reduces the optimal level of the protein �

Questions we ask…

• for each paper we will answer general and specific questions
• general questions:

6

1. What is the overarching research question (big picture)?
2. What is the specific question asked (hypothesis)?
3. How were the experiments designed (methodology)?�Which question is being answered in each figure or table? �How many experiments were performed?
4. Does the paper answer the specific question?
5. Does the paper advance the state of the field and understanding the big-picture?�What further questions and studies should be done?

Assignment before the Journal Club Session

• a WORD document that answers the questions:
• Soon after the gene that is responsible for cystic fibrosis was discovered, it was found that the gene encodes a protein that is normally found on the surface of epithelial cells. Once researchers knew this, what were all the possible hypotheses that the researchers might have entertained at that time to explain how a mutation in the gene could cause disease? List 4 hypotheses.
• What was the primary outcome in the second paper? What do you think is the most important outcome for such a study and why?

​

7

First paper: PNAS (1993)

8

1. What is the overarching research question (big picture)?

The BIG Question

9

1. What is the overarching research question (big picture)?
1. What is happening to the mutant CFTR protein such that it is not getting to the plasma membrane?
2. Where is mutant CFTR localized?

Immunoelectron microscopy: where is the CFTR?

10

A: control

B: wild type CFTR, stained with antibody

C,D: mutant CFTR, no protein on the membrane�a lot of protein in the ER

​

Immunoelectron microscopy = electron microscopy (EM) + immunological methods to visualize specific proteins or antigens within tissue (antibodies are conjugated with small electron-dense markers, often gold nanoparticles)

Where is the CFTR degraded?

11

Is the mutant CFTR degraded in lysosomes?

​

Pulse-labeling the cells with S-35 methionine (amino acid used in protein synthesis) and chased

mutant

Rapidly degrades from the ER

Ammonium chloride – the inhibitor of lysosomes (raises the PH)

No change – so CFTR is degraded not in lysosomes but somewhere else

Is CFTR degraded because it is folded?

12

Is mutant CFTR associated with chaperone proteins such as HSP70 or BiP?

Chaperone proteins help newly synthesized or denatured proteins achieve their correct three-dimensional structures

Rapidly degrades from the ER

No change – so CFTR is degraded not in lysosomes but somewhere else

• ³⁵S-Met labelling
• Immunoprecipitation

• IP with indicated Abs
• Immunoblot for CFTR

Time dependence of CFTR – HSP70 association

13

mutant CFTR does not go to the Golgi, is degraded and associates with HSP70 (chaperone protein)

Immature CFTR

After 90 min more mutant form bound to HSP70

³⁵S-Met labelling

IP CFTR

³⁵S-Met labelling

IP – Hsp70

Mutant is mostly degraded

Unanswered questions at the time

• What keeps the mutant protein trapped at the ER? - it is bound to HSP70
• How does mutant protein get degraded? – ubiquitination of the CFTR and degradation through proteasome
• Is there a way to prevent mutant CFTR from being degraded?
• If yes, can that mutant protein be trafficked to the plasma membrane and maybe become functional?

​

• Since there are at least 3 targets possible (HCP70 binding, proteasome, ubiquitination) – this is difficult to target

14

Part II

15

Pulse-chase protein labeling

• Used to study the dynamics of protein metabolism and localization
• Tagging: Sulfur-35: beta emitter (electron), half-life of 87 days

+ Methionine (amino acid) → incorporated into the newly �synthesized protein during a short “pulse” labeling period ³⁵S-methionine has the same chemical properties so it replaces the normal methionine

• “chasing”: adding an excess of unlabeled amino acids and an inhibitor of protein synthesis during a “chase” period allows one to follow the tag with precision and to establish the time course characteristic �
• Cell lysates were prepared
• with a specific antibody (anti-CFTR) then do immunoprecipitation of ³⁵S-labeled protein
• analyzed by SDS/PAGE gel electrophoresis and autoradiography

16

+

Unanswered questions at the time

• What keeps the mutant protein trapped at the ER? - it is bound to HSP70
• How does mutant protein get degraded? – ubiquitination of the CFTR and degradation through proteasome
• Is there a way to prevent mutant CFTR from being degraded?
• If yes, can that mutant protein be trafficked to the plasma membrane and maybe become functional?

​

• Since there are at least 3 targets possible (HCP70 binding, proteasome, ubiquitination) – this is difficult to target

17

Second paper

Second paper: NEJM 2019 (remember 1993)

18

2 corrector compounds

1 potentiator compound

Functional drug screening

Second paper: NEJM 2019 (remember 1993)

19

1. What is the overarching research question (big picture)?

Second paper: NEJM 2019 (remember 1993)

20

1. What is the overarching research question (big picture)?

Can a combo of 3 molecular compounds improve lung function

In CF patients with the most common F508del mutation?

Designing the trial

21

• Randomized, double-blind, placebo-controlled
• >12 years old
• Primary endpoint: absolute change in FEV₁ at week 4 (air volume that a patient can expel in 1 sec after inhalation)
• FEV1 consistent between the groups
• BMI similar (the sicker the patient the more mass they lose)
• Patient-reported quality of life similar

Results and Outcomes

22

Reduction in lung infections

improvement

The patients felt better

Putting on weight

Time evolution in response��(clinical features)

2 weeks improvement!

Patient distribution

Lower event rate for the treatment group

4 weeks improvement (losing less salt)

Widespread across

patients

Clinical improvement score – rapid jump

Time

evolution in response��(physiological defect)

Adverse effects

25

• Or no difference or lower rate for the treatment group
• Maybe more diarrhea
• Less fatigue
• No increase compared to placebo over all

Adverse effects: cost

26

• CAD 282,240 /year in Quebec

Some open questions

27

• Trikafta is a black box w.r.t fundamental mechanism of the components (correctors bringing the CFTR to the membrane and potentiators making it active)
• Late adverse effects?
• Long-term efficacy?
• How about other CF symptoms and not only pulmonary (e.g. infertility)?
• 10% of patients with other types of mutations? – unmet clinical need for a subset of CF patients