Charcot–Marie–Tooth type 2B disease-causing RAB7A mutant proteins show altered interaction with the neuronal intermediate filament peripherin

Abstract

Charcot–Marie–Tooth type 2B (CMT2B) is a peripheral ulcero-mutilating neuropathy caused by four missense mutations in the rab7a gene. CMT2B is clinically characterized by prominent sensory loss, distal muscle weakness leading to muscle atrophy, high frequency of foot ulcers and infections that often results in toe amputations. RAB7A is a ubiquitous small GTPase, which controls transport to late endocytic compartments. Although the biochemical and functional properties of disease-causing RAB7A mutant proteins have been investigated, it is not yet clear how the disease originates. To understand how mutations in a ubiquitous protein specifically affect peripheral neurons, we performed a two-hybrid screen using a dorsal root ganglia cDNA library with the purpose of identifying RAB7A interactors specific for these cells. We identified peripherin, an intermediate filament protein expressed primarily in peripheral neurons, as a putative RAB7A interacting protein. The interaction was confirmed by co-immunoprecipitation and pull-down experiments, and established that the interaction is direct using recombinant proteins. Silencing or overexpression of wild type RAB7A changed the soluble/insoluble rate of peripherin indicating that RAB7A is important for peripherin organization and function. In addition, disease-causing RAB7A mutant proteins bind more strongly to peripherin and their expression causes a significant increase in the amount of soluble peripherin. Since peripherin plays a role not only in neurite outgrowth during development but also in axonal regeneration after injury, these data suggest that the altered interaction between disease-causing RAB7A mutants and peripherin could play an important role in CMT2B neuropathy.

The Rab-interacting lysosomal protein (RILP) regulates vacuolar ATPase acting on the V1G1 subunit

“…For immunoprecipitation, we used anti-HA affinity gel (Ezview Red Anti-HA E6779, Sigma) according to the manufacturer's instructions and as described previously (Cogli et al, 2013b). Co-immunoprecipitation of endogenous proteins in HeLa cells was performed using a crosslink immunprecipitation kit (Pierce, Rockford, IL), following the manufacturer's instructions and as described previously (Cogli et al, 2013a).…”

“…Samples were then subjected to SDS-PAGE and western blotting. Pull-down experiments were as described previously (Cogli et al, 2013b). Briefly, purified His-V1G1 was bound to Ni-NTA resin and incubated with lysates of HeLa cells transfected with Glued in the presence or absence of purified GST-RILP for 2 h at 4˚C.…”

Rab7a regulates cell migration through Rac1 and vimentin

“…We demonstrated that the interaction is direct and that Rab7a regulates vimentin and peripherin filament assembly [17,18]. Overexpression of Rab7a wt or expression of the constitutively active Rab7a Q67L mutant protein increased vimentin phosphorylation and caused redistribution of vimentin in the soluble fraction, while, coherently, Rab7a-depletion determined a decreased phosphorylation of vimentin and increased the amount of filamentous vimentin [17,18].…”

“…Importantly, the small GTPase Rab7a regulates the assembly of vimentin affecting also its phosphorylation state [17,18,74]. Interestingly, in migrating cells, Rab7a-positive vesicles localize in the perinuclear region facing the wound while vimentin filaments are reorganized and reoriented in the direction of the wound (Fig.…”

“…Recently, we discovered that Rab7a interacts also with two members of the intermediate filament proteins, vimentin and peripherin [17,18]. We demonstrated that the interaction is direct and that Rab7a regulates vimentin and peripherin filament assembly [17,18].…”