Characterization of the Rab7K157N mutant protein associated with Charcot–Marie-Tooth type 2B Azzurra De Luca, Cinzia Progida, Maria Rita Spinosa, Pietro Alifano, Cecilia Bucci * Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, Via Provinciale Monteroni, 73100 Lecce, Italy article info Article history: Received 5 May 2008 Available online 21 May 2008 Keywords: Rab7 Rab proteins Charcot–Marie-Tooth CMT2B Axonal neuropathy Neurodegeneration Axon degeneration Endocytosis EGF degradation Peripheral neuropathies abstract Four missense mutations, that target highly conserved amino acid residues in the small GTPase Rab7, have been associated with the Charcot–Marie-Tooth (CMT) type 2B phenotype. CMT2B peripheral axonal neuropathies are characterized by severe sensory loss, often complicated by infections, arthropathy, and amputations. Here, we have investigated the biochemical and functional properties of the Rab7 K157N mutated protein. Interestingly, Rab7 K157N showed altered nucleotide exchange rate and GTP hydrolysis compared to the wild type protein. Consistently, the majority of the expressed protein in HeLa cells was bound to GTP. In addition, Rab7 K157N was able to restore EGF degradation, previously inhibited by Rab7 silencing. Altogether these data indicate that Rab7 K157N, similarly to the other three mutated proteins causative of CMT2B, is predominantly in the GTP-bound form and behaves as an active mutant. Therefore, activated forms of Rab7 protein cause the CMT2B disease. Ó 2008 Elsevier Inc. All rights reserved. Hereditary neuropathies of the peripheral nervous system are genetically and clinically heterogeneous and affect neurons and/ or Schwann cells [1–6]. Charcot–Marie-Tooth (CMT) disease is the commonest inherited neuromuscular disorder affecting one in 2500. The disease is due to mutations in several different genes that cause similar phenotypes. Indeed, since 1991 over 40 causa- tive genes have been described and some of them are clearly asso- ciated with mitochondria or membrane traffic. Historically, CMT is classified mainly into type 1 (CMT1) and type 2 (CMT2) forms. CMT1 is a dominant inherited demyelinating neuropathy charac- terized by reduced nerve conduction velocities while CMT2 is a dominantly inherited axonal neuropathy characterized by normal or slightly reduced nerve conduction velocities. CMT2 is defined axonal since it shows chronic axonal degeneration with loss of nerve fibers. Besides these main classes more rare forms are also included in CMT. Among CMT2 forms, the CMT2 type B (CMT2B) is clinically characterized by marked distal muscle weakness, high frequency of foot ulcers, infections, and amputations of the toes be- cause of recurrent infections. Four missense mutations that target highly conserved amino acid residues in the GTP binding and hydrolysis domains of the small GTPase Rab7 have been identified as causative of CMT2B [7–9]. Rab proteins regulate membrane traffic by recruiting effec- tor proteins with different functions [10]. Rab7, a small GTPase of the Rab family, regulates transport to endocytic degradative compartments. Indeed, Rab7 is a key protein for the maturation of lysosomes, phagolysosomes, and autophagosomes but also for growth factor-regulated cell nutrition and apoptosis [11–16]. Re- cent studies have indicated that Rab7 controls retrograde axonal transport of neurotrophin receptors [17,18]. As internalization and retrograde axonal transport of neurotrophin receptors are essential for neurotrophin signaling and control neuronal differen- tiation, plasticity, and survival, altered neurotrophin trafficking could heavily influence neurodegeneration [19,20]. Therefore, CMT2B disease could be easily explained if the identified muta- tions in the Rab7 protein impair Rab7 function determining altered neurotrophin trafficking. Recently, we have analyzed the biochemical properties of three of the four mutant proteins causing CMT2B, namely Rab7 L129F, Rab7 N161T, and Rab7 V162M [21]. We have discovered that all three mutant proteins have altered nucleotide binding and hydro- lysis activity, suggesting the same mechanism of action [21]. In this study, we characterize the biochemical properties of the fourth mutant protein (Rab7 K157N) causative of CMT2B. We dem- onstrated that, similarly to the other three mutated proteins, Rab7 K157N has reduced affinity for GDP and impaired hydrolysis activ- ity, and it behaves as an active form of the Rab7 protein. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.060 * Corresponding author. Fax: +39 0832 298626. E-mail address: cecilia.bucci@unile.it (C. Bucci). Biochemical and Biophysical Research Communications 372 (2008) 283–287 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc