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Involvement of zDHHC 5, 7, 8 in the formation of the retromer in endosomal-to-TGN trafficking
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Involvement of zDHHC 5, 7, 8 in the formation of the retromer in endosomal-to-TGN trafficking
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Involvement of zDHHC 5, 7, 8 in the formation of the retromer in endosomal-to-TGN trafficking
Proper binding of the retromer complex to the endosome is known to be crucial for accurate and
efficient trafficking of cargo within the endosome-to-TGN pathway. Dysfunction in this complex has been
shown to be crucial in the development of neurodegenerative diseases, like Parkinson’s and Alzheimer’s.
The palmitoylation of Rab7 is needed to allow this binding and is achieved by the action of the zDHCC-
PAT family. Understanding which within this family is crucial for a better understanding of cargo
trafficking as a whole. Three DHHCs have been found to be localized or play a role within the trafficking
pathway, with DHHC5 showing the greatest potential. The main objective of our study is to identify the
involvement of these zDHHCs in the palmitoylation of the Rab required in the formation of the retromer
complex to the endosome. To investigate this we will use HeLa cell cultures, siRNA’s, western blot,
antibody retrieval assays, and two-color STORM with fluorescently linked antibodies. A greater
understanding of which DHHC is involved in the palmitoylation of Rab7, allowing for further insight and
understanding of retromer binding and possibly its restoration.
Introduction
Retromer, a conserved complex of proteins that have been shown to be crucial for transferring
endocytosed proteins from endosomes to the plasma membrane or the trans-Golgi network (TGN), via recycling
mechanisms [1]. It is among others in charge of retrieving cargo receptors from the endosomes to the TGN and
recruiting proteins from endomes immediately back to the plasma membrane [1,2]. The complex includes two
big parts, namely the sorting nexin proteins (SNX proteins) and a Vps26-Vps29-Vps35 trimmer, also called the
cargo recognition complex (CRC). As retromer is crucial for the maintenance of neurons, dysfunction of this
complex has been shown to be involved in neurodegenerative diseases e.g. Parkison’s disease and Alzheimer’s
disease [1].
To recruit a retromer from the endosomes, Rab7 is necessary [2]. It is also demonstrated that Rab7 is
analyzed for palmitoylated neuronal proteins [2]. During palmitoylation, i.e. S-Acylation (fig. 1), a palmitate
group typically from palmitoyl-CoA is able to attach to a cysteine side of a protein, through a reversible
thioester bond [3]. Palmitoylation can adjust the activity of a protein in different ways, for instance in
membrane binding, sorting, localization, and stability. Enzymes, i.e. Palmitoyltransferases (PATS), mediate this
post-translational modification, consisting of a conserved catalytic area, marked by the amino acid sequence
aspartic acid-histidine-histidine-cysteine (zDHHC) [3]. It is known that a few proteins can be s-palmitoylated
by different DHHC, while others require the action of a specific enzyme [3].
Figure 1: The S-
acylation pathway of
a substrate (Rab7)
through the
enzymatic activity of
zDHHC-PATs
broken down into
consequential steps,
as described by Jin et al. [4].
As of now, it is not clear what the substrate specificity is of each individual DHHC. The zDHHC-PATs
family represents multigene families, which means the individual members have a very high level of sequence
similarities [5]. This poses a great challenge in the efforts to assign specific substrates to individual DHHCs [5].
Our main objective is to gain further insight on which members of the CDCDHHC-PATs family are
involved in the s-palmitoylation of the Rab. Specifically, zDHHC 5,7, and 8 will be the focus of this assay. The
reason for this is that zDHHC 5 has been found to play a role in endosome-to-TGN trafficking [6]. The choice
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