ABSTRACT
Title
Hereditary spastic paraplegia protein REEP1: molecular and functional studies
Authors
V De Nardo
Doctorate School in Molecular and Cellular Pharmacology
Dept. of Pharmacology and Anesthesiology – University of Padova, Italy
Doctorate School in Molecular and Cellular Pharmacology
Dept. of Pharmacology and Anesthesiology – University of Padova, Italy
Abstract
Hereditary spastic paraplegias (HSPs) comprise a group of inherited neurological disorders with the cardinal feature of progressive spasticity and weakness of the lower extremities, due to a lengthdependent axonopathy of corticospinal motor neurons [1–5]. HSPs have historically been classified as pure if spastic paraparesis occurs in isolation and complicated if other neurological abnormalities are present [6]. More recently, a molecular genetic classification has come into wide use, with over 40 different genetic loci (SPG1–45) reported [7]. The identification of genes for 20 of these HSPs has stimulated development of a classification scheme based on possible pathogenic mechanisms. These include mitochondrial dysfunction, abnormalities in axonal pathfinding or myelination, and intracellular trafficking defects [1–5]. Despite the daunting number of distinct genetic loci, well over 50% of HSP patients harbor pathogenic mutations in 1 of just 3 genes: spastin (SPG4, also known as SPAST), atlastin-1 (SPG3A, also known as ATL1), or receptor expression enhancing protein 1(REEP1, also known as SPG31).
In this study we investigated the distribution and function of REEP1 protein.
REEP1 gene consists of seven exons and codifies for a protein of 202 amino acids with a molecular weight of 22.25 kDa. REEP1 contains two predicted transmembrane domains (TM1 and TM2) and a domain of unknown function called “deleted in polyposis” (TB2/DP1/HVA22).
The first part of this project was focused on the generation of different REEP1 constructs with HA and MYC fused to the N-terminal and C-terminal part of the protein to study the expression profile and the topology of REEP1.
REEP1 protein was overexpressed in HeLa and Cos7 cells by transfection and the cells were analyzed by immunofluorescence staining. This experiment suggest that REEP1 is localized on the endoplasmic reticulum (ER) membrane and it’s mostly reticular; we also analyzed the orientation of REEP1 in the lipid bilayer of ER by a selective permeabilization of cellular membranes using digitonin (20μM) and Triton X100 (1%) and we discovered that both domains (C-terminal and N-terminal) of REEP1 are orientated to the cytoplasm. We confirm this result by immunoblotting, after a selective precipitation of the cellular components.
Moreover, to study the pathological mechanism of HSP, we have reproduced in the wild type REEP1 protein two pathological mutations found in patients: A20E and P19R.
REEP1 P19R mutation, byimmunofluorescence experiments, appeared less reticular than wild type protein, and it presents manyvesicles that we discovered there are localized around lipid droplets: lipid droplets are the lipid storage organelles of all organisms; for a long time it was thought that lipid droplets only act as storage depots. More recent data support the idea that lipid droplets are highly dynamic organelles which participate in several cellular processes and interact with various other cellular compartments [8]. Thesefindingssuggestthat REEP1 is involved in the metabolism of fatty acids and in particularly in the trafficking of lipid droplets.
REEP1 A132V mutation surprisingly isn’t localized on the endoplasmic reticulum but almost co-localizedwith microtubules. We know by literature that the C terminal part of REEP1 is involved in interaction with microtubules [9]; also lipid droplets moving from endoplasmic reticulum to the other cellular compartments by microtubules. This evidence support the hypothesis that REEP1 is involved in lipid droplets trafficking.
References:
1) Reid E (2003) J Med Genet 40:81-86
2) Fink JK (2006) Curr Neurol Neurosci Rep 6:65-76
3) Soderblom C et al. (2006) Pharmacol Ther. 109:42-56
4) Zuchner S (2007) Expert Opin Pharmacother 8:1433-1439
5) Salinas S et al. (2008) Lancet Neurol 7:1127-1138
6) Harding AE (1983) Lancet 1:1151-1155
7) Durr A (2008) Neurology 71:236-238
8) Beller M (2008) Plos Biol 6:292
9) Seong H (2010) J Clin Invest 120:1097-110
In this study we investigated the distribution and function of REEP1 protein.
REEP1 gene consists of seven exons and codifies for a protein of 202 amino acids with a molecular weight of 22.25 kDa. REEP1 contains two predicted transmembrane domains (TM1 and TM2) and a domain of unknown function called “deleted in polyposis” (TB2/DP1/HVA22).
The first part of this project was focused on the generation of different REEP1 constructs with HA and MYC fused to the N-terminal and C-terminal part of the protein to study the expression profile and the topology of REEP1.
REEP1 protein was overexpressed in HeLa and Cos7 cells by transfection and the cells were analyzed by immunofluorescence staining. This experiment suggest that REEP1 is localized on the endoplasmic reticulum (ER) membrane and it’s mostly reticular; we also analyzed the orientation of REEP1 in the lipid bilayer of ER by a selective permeabilization of cellular membranes using digitonin (20μM) and Triton X100 (1%) and we discovered that both domains (C-terminal and N-terminal) of REEP1 are orientated to the cytoplasm. We confirm this result by immunoblotting, after a selective precipitation of the cellular components.
Moreover, to study the pathological mechanism of HSP, we have reproduced in the wild type REEP1 protein two pathological mutations found in patients: A20E and P19R.
REEP1 P19R mutation, byimmunofluorescence experiments, appeared less reticular than wild type protein, and it presents manyvesicles that we discovered there are localized around lipid droplets: lipid droplets are the lipid storage organelles of all organisms; for a long time it was thought that lipid droplets only act as storage depots. More recent data support the idea that lipid droplets are highly dynamic organelles which participate in several cellular processes and interact with various other cellular compartments [8]. Thesefindingssuggestthat REEP1 is involved in the metabolism of fatty acids and in particularly in the trafficking of lipid droplets.
REEP1 A132V mutation surprisingly isn’t localized on the endoplasmic reticulum but almost co-localizedwith microtubules. We know by literature that the C terminal part of REEP1 is involved in interaction with microtubules [9]; also lipid droplets moving from endoplasmic reticulum to the other cellular compartments by microtubules. This evidence support the hypothesis that REEP1 is involved in lipid droplets trafficking.
References:
1) Reid E (2003) J Med Genet 40:81-86
2) Fink JK (2006) Curr Neurol Neurosci Rep 6:65-76
3) Soderblom C et al. (2006) Pharmacol Ther. 109:42-56
4) Zuchner S (2007) Expert Opin Pharmacother 8:1433-1439
5) Salinas S et al. (2008) Lancet Neurol 7:1127-1138
6) Harding AE (1983) Lancet 1:1151-1155
7) Durr A (2008) Neurology 71:236-238
8) Beller M (2008) Plos Biol 6:292
9) Seong H (2010) J Clin Invest 120:1097-110