Explorer l'impact de l'élimination de la charge nette cationique des signaux de localisation nucléaire (NLS) sur le ciblage par l'importine-α

The nucleus is the control center of the eukaryotic cells as it contains most of the genetic material (DNA) necessary for host survival. As the DNA and DNA-interacting proteins in the nucleus are highly sensitive to damage, the nucleus is a target for therapeutic intervention for many diseases such as cancer. However, the nucleus is protected by a double membrane that only allows low molecular weight molecules to freely diffuse through nuclear pores, making it challenging to target. Nuclear transport is a vital component of normal functioning in eukaryotic cells. Specialized transport proteins called importins recognize nuclear localization signals (NLSs) on proteins in the cytoplasm requiring nuclear entry. This biological phenomenon has spurred extensive research into NLS-tagged therapeutics to 8actively9 target the nucleus to maximize therapeutic efficacy. Despite many advancements in NLS-therapeutic design, the absence of consistent successes within clinical settings prompts critical questions about the factors contributing to this challenge. Addressing this question drives the rational of this thesis. Typically, NLSs are highly cationic nature due to the importance that lysine and arginine amino acids are required to form crucial contacts with the side chains of importin-ɑ in the major or minor NLS-binding pockets. From a pharmaceutical perspective, it is known that therapeutic agents with increased net-positive molecular charges exhibit poor pharmacokinetics due to significant non-specific ionic interactions with mammalian cell membranes that are rich in anionic phospholipids. As a result, when these agents are administered intravenously, they are rapidly eliminated from the blood stream and insufficiently accumulate at the tumor site to evoke effective therapeutic efficacy. Labeling NLSs to therapeutics requires high NLS-to-antibody ratios required to achieve efficient nuclear import. In the current work, we have designed and developed novel NLSs bearing zero net-charge. This thesis examines the importin-ɑ ability of these net zero NLSs using experimental methods concentrating on biophysical and biochemical techniques. Specifically, crystal structures were obtained with the net zero NLSs complexed to importin-ɑ. Circular dichroism and microscale thermophoresis were employed to evaluate NLS-importin-ɑ complex stabilities and binding affinities. Our results showed that zero net-charge NLSs could be developed with binding affinities and interaction modes comparable to those of natural NLSs. The anionic amino acids in the net zero NLSs formed novel contacts with importin-ɑ, while maintaining the same binding modes as the natural NLS counterparts. Taken together, this research opens the door for the development of more effective NLS-based therapies with potentially improved pharmacokinetics and increased tumor-site accumulation.

Name thesis:degree_name

M. Sc.

Level thesis:degree_level

Maîtrise

Discipline thesis:degree_discipline

Sciences des radiations et imagerie biomédicale

Grantor dc:publisher

Université de Sherbrooke

Year dc:date.issued

2024

Author dc:creator

• Abdoli, Amirabbas

Advisors dc:contributor.advisor

• Leyton, Victor Jeffrey
• Lavigne, Pierre

• NLS
• Importin-ɑ
• Zero net-charge
• MST
• Circular dichroism
• TAF8

Language dc:language.iso

fr, en

Handle dc:identifier.uri

http://hdl.handle.net/11143/21828

OAI identifier oai:identifier

oai:usherbrooke.scholaris.ca:11143/21828