BADERC-Vladimir Marshansky

Vladimir Marshansky, M.D., Ph.D.

V-ATPase/smallGTPases/ARNO/aldolase complex and regulation of protein degradative pathway

Our research project is driven by recent novel discoveries that: 1) V-ATPase (vacuolar-type H+-ATPase) is an endosomal pH-sensor [1-8] which directly interacts with Arf6 small GTPase and with critical regulatory elements of the Arf-GEF, ARNO (ADP-ribosylation factor nucleotide site opener) [9-11]; 2) ARNO also directly interacts with aldolase, a central enzyme of the glycolytic pathway which in turn is crucial for regulation of V-ATPase function [2-4]; 3) these interactions regulate the endosomal/lysosomal protein degradative pathway in kidney proximal tubule (PT) cells [1-3]. Here we propose: 1) that V-ATPase may not simply recruit and scaffold small GTPases to their target membranes but could also, via its interaction with regulatory elements in ARNO, have a novel role as an activator of Arf-GEF activity and therefore of Arf-amily small GTPases; 2) that V-ATPase/small GTPase/ARNO/aldolase complex plays a key role in glucose-dependent regulation of V-ATPase function, which could have profound consequences in diabetes, a disease associated with high levels of plasma glucose.

Our main hypothesis is that high glucose levels during the early stages of diabetes could modulate V-ATPase/small GTPase/ARNO/aldolase interactions and modulate acidification of endosomal/lysosomal compartments of the protein degradative pathway. This, in turn, leads to aberrant vesicular trafficking and abnormal protein degradation. Thus, the main goals of our project are: 1) to determine the molecular mechanism of interaction and regulation of ARNO by V-ATPase; 2) to uncover the molecular mechanism and cell biological significance of the interaction between ARNO and aldolase; 3) to elucidate the molecular basis and biological significance of the V-ATPase/small GTPase/ARNO/aldolase complex in regulation of the endosomal/ lysosomal protein degradative pathway of kidney proximal tubules during early stages of Type I diabetes. These studies are performed using non-obese diabetic (NOD) mice as a model of human Type I diabetes and mouse tubular cells (MTC) exposed to high levels of glucose. While the late stages of diabetes (months/years) leading to end-stage renal disease (ESRD) are well studied, important early (1-4 weeks) events that initiate this pathophysiological process remain unknown. Abnormal function of the protein-degradative pathway may have important implications for the pathophysiology of diabetes in general and in development of early stages of diabetic nephropathy in particular. Thus our project represents a directed search for early cell biological events that could provide potential therapeutic targets for prevention of diabetic nephropathy.

References:

1. Hurtado-Lorenzo A., Skinner M., El Annan J., Futai M., Sun-Wada GH, Bourgoin S., Casanova J., Wildeman A., Bechoua S., Ausiello D., Brown D., Marshansky V. V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. – Nature Cell Biology, (2006) 8,124-136.

2. Marshansky V. The V-ATPase a2-subunit as a putative endosomal pH-sensor. – Biochem Soc Trans (2007) 35, 1092-1099.

3. Marshansky V. Futai M. The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function. –Curr Opin Cell Biol (2008) 20, 415-426.

4. Grüber G, Marshansky V. New insights into structure-function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0). – BioEssays (2008) 30, 1096 -1099.

5. Merkulova M., Marshansky V. Function of V-ATPase as a putative endosomal pH-sensor: mapping the interaction sites between V-ATPase a2-isoform and ARNO. Abstract Experimental Biology Meeting (San Diego) – The FASEB Journal (2008) 22, 11563.

6. Brown D., Breton S., Ausiello DA., Marshansky V. Sensing, signaling and sorting events in kidney epithelial cell physiology. – Traffic (2009)(In Press)

7. Brown D., Paunescu TG., Breton S., Marshansky V. Regulation of the V-ATPase in kidney epithelial cells: dual role in acid/base homeostasis and vesicular trafficking. – J. Exp. Biol. (2009) (In Press)

8. Merkulova M., Bakulina A., Thaker Y.R., Gruber G, Marshansky V. Specific domains on the V-ATPase a2-isoform interact with regulatory elements of ARNO – J Biol Chem (2009) (Submitted after Revision)

9. Merkulova M., Hurtado-Lorenzo A., Brown D., Ausiello D.A., Marshansky V. V-ATPase/small GTPase/ aldolase complex and regulation of endosomal/lysosomal protein degradative pathway. – Experimental Biology Meeting (New Orleans, 2009) Abstract.

10. Marshansky V., Merkulova M. Structural and functional aspects of V-ATPase/small GTPase/ ARNO/aldolase complex: potential role in regulation of protein degradation pathway during diabetes. – Gordon Conference “Molecular and Cellular Bioenergetics: From Crystal Structure to Human Disease”, (Proctor Academy, Andover, NH, 2009) Abstract.

11. Merkulova M., Hurtado-Lorenzo A., Brown D., Ausiello D.A., Marshansky V. Proteomic analysis of ARNO interactome: Direct interaction with aldolase B and a-subunit isoforms of V-ATPase. – Mol & Cell Proteomics (2009) (Submitted)






			Vladimir Marshansky, M.D., Ph.D. V-ATPase/smallGTPases/ARNO/aldolase complex and regulation of protein degradative pathway Our research project is driven by recent novel discoveries that: 1) V-ATPase (vacuolar-type H+-ATPase) is an endosomal pH-sensor [1-8] which directly interacts with Arf6 small GTPase and with critical regulatory elements of the Arf-GEF, ARNO (ADP-ribosylation factor nucleotide site opener) [9-11]; 2) ARNO also directly interacts with aldolase, a central enzyme of the glycolytic pathway which in turn is crucial for regulation of V-ATPase function [2-4]; 3) these interactions regulate the endosomal/lysosomal protein degradative pathway in kidney proximal tubule (PT) cells [1-3]. Here we propose: 1) that V-ATPase may not simply recruit and scaffold small GTPases to their target membranes but could also, via its interaction with regulatory elements in ARNO, have a novel role as an activator of Arf-GEF activity and therefore of Arf-amily small GTPases; 2) that V-ATPase/small GTPase/ARNO/aldolase complex plays a key role in glucose-dependent regulation of V-ATPase function, which could have profound consequences in diabetes, a disease associated with high levels of plasma glucose. Our main hypothesis is that high glucose levels during the early stages of diabetes could modulate V-ATPase/small GTPase/ARNO/aldolase interactions and modulate acidification of endosomal/lysosomal compartments of the protein degradative pathway. This, in turn, leads to aberrant vesicular trafficking and abnormal protein degradation. Thus, the main goals of our project are: 1) to determine the molecular mechanism of interaction and regulation of ARNO by V-ATPase; 2) to uncover the molecular mechanism and cell biological significance of the interaction between ARNO and aldolase; 3) to elucidate the molecular basis and biological significance of the V-ATPase/small GTPase/ARNO/aldolase complex in regulation of the endosomal/ lysosomal protein degradative pathway of kidney proximal tubules during early stages of Type I diabetes. These studies are performed using non-obese diabetic (NOD) mice as a model of human Type I diabetes and mouse tubular cells (MTC) exposed to high levels of glucose. While the late stages of diabetes (months/years) leading to end-stage renal disease (ESRD) are well studied, important early (1-4 weeks) events that initiate this pathophysiological process remain unknown. Abnormal function of the protein-degradative pathway may have important implications for the pathophysiology of diabetes in general and in development of early stages of diabetic nephropathy in particular. Thus our project represents a directed search for early cell biological events that could provide potential therapeutic targets for prevention of diabetic nephropathy. References: 1. Hurtado-Lorenzo A., Skinner M., El Annan J., Futai M., Sun-Wada GH, Bourgoin S., Casanova J., Wildeman A., Bechoua S., Ausiello D., Brown D., Marshansky V. V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. – Nature Cell Biology, (2006) 8,124-136. 2. Marshansky V. The V-ATPase a2-subunit as a putative endosomal pH-sensor. – Biochem Soc Trans (2007) 35, 1092-1099. 3. Marshansky V. Futai M. The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function. –Curr Opin Cell Biol (2008) 20, 415-426. 4. Grüber G, Marshansky V. New insights into structure-function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0). – BioEssays (2008) 30, 1096 -1099. 5. Merkulova M., Marshansky V. Function of V-ATPase as a putative endosomal pH-sensor: mapping the interaction sites between V-ATPase a2-isoform and ARNO. Abstract Experimental Biology Meeting (San Diego) – The FASEB Journal (2008) 22, 11563. 6. Brown D., Breton S., Ausiello DA., Marshansky V. Sensing, signaling and sorting events in kidney epithelial cell physiology. – Traffic (2009)(In Press) 7. Brown D., Paunescu TG., Breton S., Marshansky V. Regulation of the V-ATPase in kidney epithelial cells: dual role in acid/base homeostasis and vesicular trafficking. – J. Exp. Biol. (2009) (In Press) 8. Merkulova M., Bakulina A., Thaker Y.R., Gruber G, Marshansky V. Specific domains on the V-ATPase a2-isoform interact with regulatory elements of ARNO – J Biol Chem (2009) (Submitted after Revision) 9. Merkulova M., Hurtado-Lorenzo A., Brown D., Ausiello D.A., Marshansky V. V-ATPase/small GTPase/ aldolase complex and regulation of endosomal/lysosomal protein degradative pathway. – Experimental Biology Meeting (New Orleans, 2009) Abstract. 10. Marshansky V., Merkulova M. Structural and functional aspects of V-ATPase/small GTPase/ ARNO/aldolase complex: potential role in regulation of protein degradation pathway during diabetes. – Gordon Conference “Molecular and Cellular Bioenergetics: From Crystal Structure to Human Disease”, (Proctor Academy, Andover, NH, 2009) Abstract. 11. Merkulova M., Hurtado-Lorenzo A., Brown D., Ausiello D.A., Marshansky V. Proteomic analysis of ARNO interactome: Direct interaction with aldolase B and a-subunit isoforms of V-ATPase. – Mol & Cell Proteomics (2009) (Submitted)