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Identification of changes in the de novo proteome underlying the strengthening of synaptic connections

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

How do some memories last? The long-lasting strengthening of synaptic connections between neurons, also known as long-term potentiation (LTP), is thought to be the main cellular phenomenon underlying our capacity to form memories. To sustain LTP for a long period of time, a group of proteins, termed plasticity-related proteins (PRPs), is thought to be synthesized and transported to synapses. Identifying PRPs requires techniques of high temporal resolution.
I first used tandem mass tagging (TMT) technique to examine global proteomic changes after in vivo LTP induction in the hippocampus of anesthetized rats, and after in vitro chemical LTP (cLTP) induction in rat organotypic hippocampal slice cultures. Without the adjustment for false discovery rate, the MS data showed significant differential regulation of 237 proteins after LTP induction in vivo and 83 proteins after cLTP induction in vitro. Gene ontology analysis and literature review of the identified candidates proposed SV2-related protein (SVOP), constitutive photomorphogenesis 9 (COP9), and protein phosphatase 1 regulatory subunit 29 (ELFN2) as interesting candidates for further research.
To increase the temporal resolution and specifically examine the nascent proteome, I utilized a novel combinatorial chemoproteomic technique known as BONCAT-TMT, which combines bioorthogonal non-canonical amino acid tagging (BONCAT) and tandem mass tagging (TMT) of proteins. I used BONCAT-TMT to perform multiplexed purification, identification and quantification of proteins that are newly synthesized upon induction of cLTP in vitro in organotypic hippocampal slice cultures. Preliminary data from BONCAT-TMT shows that newly synthesized components of the ubiquitin-proteasome system undergo the greatest increase after cLTP induction in vitro. However, more replicates are required before such changes can be deemed reproducible or tested for statistical relevance.
To both enhance and make the most use of the temporal resolution of BONCAT, several photocaged synthetic amino acid derivatives were developed that exhibit successful light-activated labelling of newly synthesized proteins (NSPs) in cell culture. Preliminary data shows that 7-nitroindoline and coumarin photocaged derivatives of azidohomoalanine (AHA) are capable of labelling NSPs in HEK293 cells after uncaging with UV light.
In an attempt to enhance AHA’s membrane permeability, AHA was esterified into methyl AHA (M-AHA) and acetoxymethyl AHA (AM-AHA). Preliminary data shows that M-AHA and AM-AHA do not exhibit higher labelling intensity of NSPs than free unconjugated AHA in HEK293 cells.
Altogether, this study is the first to explore changes in the de novo proteome after LTP induction in vitro and it is one of the first studies to develop photocaged probes that can be used to examine the nascent proteome with temporal control.
OriginalsprogEngelsk
ForlagAarhus Universitet
Antal sider147
StatusUdgivet - jun. 2021

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