CMT 2B: Functional Characterization of Rab7 Mutant Proteins Associated with Char

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The Journal of Neuroscience, February 13, 2008, 28(7):1640-1648

Functional Characterization of Rab7 Mutant Proteins Associated with

Charcot-Marie-Tooth Type 2B Disease

Rita Spinosa, * Cinzia Progida, * Azzurra De Luca,

ria Colucci, Pietro Alifano, and Cecilia Bucci

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali,

Università del Salento, 73100 Lecce, Italy

Charcot-Marie-Tooth (CMT) type 2 neuropathies are a group of

autosomal-dominant axonal disorders genetically and clinically

heterogeneous. In particular, CMT type 2B (CMT2B) neuropathies are

characterized by severe sensory loss, often complicated by

infections, arthropathy, and amputations. Recently, four missense

mutations in the small GTPase Rab7 associated with the Charcot-Marie

Tooth type 2B phenotype have been identified. These mutations target

highly conserved amino acid residues.

However, nothing is known about whether and how these mutations

affect Rab7 function. We investigated the biochemical and functional

properties of three of the mutant proteins. Interestingly, all three

proteins exhibited higher nucleotide exchange rates and hydrolyzed

GTP slower than the wild-type protein. In addition, whereas 23% of

overexpressed wild-type Rab7 was GTP bound in HeLa cells, the large

majority of the mutant proteins (82–89%) were in the GTP-bound form,

consistent with the data on GTP hydrolysis and exchange rates.

The CMT2B-associated Rab7 proteins were also able to bind the Rab7

effector RILP (Rab-interacting lysosomal protein) and to rescue Rab7

function after silencing. Altogether, these data demonstrate that all

tested CMT2B-associated Rab7 mutations are mechanistically similar,

suggesting that activated forms of the Rab7 are responsible for CMT2B

disease.