The Experts below are selected from a list of 22071 Experts worldwide ranked by ideXlab platform
Angela Wandingerness - One of the best experts on this subject based on the ideXlab platform.
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Rab7 mutants associated with charcot marie tooth disease cause delayed growth factor receptor transport and altered endosomal and nuclear signaling
Journal of Biological Chemistry, 2013Co-Authors: Soumik Basuray, Sanchita Mukherjee, Elsa Romero, Matthew N J Seaman, Angela WandingernessAbstract:Rab7 belongs to the Ras superfamily of small GTPases and is a master regulator of early to late endocytic membrane transport. Four missense mutations in the late endosomal Rab7 GTPase (L129F, K157N, N161T, and V162M) cause the autosomal dominant peripheral neuropathy Charcot-Marie-Tooth type 2B (CMT2B) disease. As yet, the pathological mechanisms connecting mutant Rab7 Protein expression to altered neuronal function are undefined. Here, we analyze the effects of Rab7 CMT2B mutants on epidermal growth factor (EGF)-dependent intracellular signaling and trafficking. Three different cell lines expressing Rab7 CMT2B mutants and stimulated with EGF exhibited delayed trafficking of EGF to LAMP1-positive late endosomes and lysosomes and slowed EGF receptor (EGFR) degradation. Expression of all Rab7 CMT2B mutants altered the Rab7 activation cycle, leading to enhanced and prolonged EGFR signaling as well as variable increases in p38 and ERK1/2 activation. However, due to reduced nuclear translocation of p38 and ERK1/2, the downstream nuclear activation of Elk-1 was decreased along with the expression of immediate early genes like c-fos and Egr-1 by the disease mutants. In conclusion, our results demonstrate that Rab7 CMT2B mutants impair growth factor receptor trafficking and, in turn, alter p38 and ERK1/2 signaling from perinuclear, clustered signaling endosomes. The resulting down-regulation of EGFR-dependent nuclear transcription that is crucial for normal axon outgrowth and peripheral innervation offers a crucial new mechanistic insight into disease pathogenesis that is relevant to other neurodegenerative diseases.
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rab 7 an important regulator of late endocytic membrane traffic
Journal of Cell Biology, 1995Co-Authors: Yan Feng, Barry Press, Angela WandingernessAbstract:Rab5 and Rab7 Proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 Protein plays an important regulatory role in early endocytosis, yet the function of Rab7 Protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G Protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the Rab7 Protein (Rab7N125I and Rab7T22N). Overexpression of wild-type Rab7 Protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the Rab7N125I mutant caused the VSV G Protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that Rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant Rab7N125I or Rab7T22N Proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN Protein. In contrast, the mutant Proteins markedly inhibited the subsequent cleavage of the SV5 HN Protein. Taken together, these data support a key role for Rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.
Cecilia Bucci - One of the best experts on this subject based on the ideXlab platform.
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role of the small gtpase Rab7 in the late endocytic pathway
Journal of Biological Chemistry, 1997Co-Authors: Rosalba Vitelli, Daniela Lattero, Maurizio Bifulco, Mario Chiariello, Mariarosaria Santillo, Carmelo B. Bruni, Cecilia BucciAbstract:Abstract Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoProteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 Protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.
Rosalba Vitelli - One of the best experts on this subject based on the ideXlab platform.
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role of the small gtpase Rab7 in the late endocytic pathway
Journal of Biological Chemistry, 1997Co-Authors: Rosalba Vitelli, Daniela Lattero, Maurizio Bifulco, Mario Chiariello, Mariarosaria Santillo, Carmelo B. Bruni, Cecilia BucciAbstract:Abstract Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoProteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 Protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.
Yan Feng - One of the best experts on this subject based on the ideXlab platform.
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rab 7 an important regulator of late endocytic membrane traffic
Journal of Cell Biology, 1995Co-Authors: Yan Feng, Barry Press, Angela WandingernessAbstract:Rab5 and Rab7 Proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 Protein plays an important regulatory role in early endocytosis, yet the function of Rab7 Protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G Protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the Rab7 Protein (Rab7N125I and Rab7T22N). Overexpression of wild-type Rab7 Protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the Rab7N125I mutant caused the VSV G Protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that Rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant Rab7N125I or Rab7T22N Proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN Protein. In contrast, the mutant Proteins markedly inhibited the subsequent cleavage of the SV5 HN Protein. Taken together, these data support a key role for Rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.
Herbert Waldmann - One of the best experts on this subject based on the ideXlab platform.
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Intein-mediated synthesis of geranylgeranylated Rab7 Protein in vitro
Journal of the American Chemical Society, 2002Co-Authors: Kirill Alexandrov, Ines Heinemann, Thomas Durek, Vadim Sidorovitch, Roger S. Goody, Herbert WaldmannAbstract:Production of recombinant Proteins is an important prerequisite for biotechnology and life sciences in general. However, there is a paucity of methods for production of posttranslationally modified recombinant Proteins or Proteins with non-native functional groups, such as fluorophores, spin labels, and so forth. In this work we have used a combination of organic synthesis and in vitro Protein ligation to construct monoprenylated Rab7 GTPase. The Protein was prepared from a recombinant N-terminal portion and a peptide mimicking the C terminus of Rab7. For construction of a synthetic six-amino-acid-long fluorescent monoprenylated peptide, we used a block condensation strategy. Ligation was achieved with a yield of >70%. The resulting Protein was purified from the unligated peptide by a combination of organic extraction and phase partitioning and refolding. The refolded monoprenylated semisynthetic Rab7 Protein (Rab7GG) formed a stable complex with its natural chaperone REP-1 (Rab escort Protein 1) and could serve as an acceptor of the second prenyl group in the enzymatic prenylation reaction. Using fluorescence spectroscopy, we characterized the interaction of the Rab7GG:REP-1 complex with Rab geranylgeranyl transferase and came to the conclusion that it functioned as a genuine intermediate of the prenylation reaction. Thus, we present the first example of the in vitro generation of a semisynthetic lipidated Protein using the native chemical ligation method.
