GSNOR/S-nitrosothiol Regulation of Vesicular Transport | Dr. Lisa Palmer, PhD

October 7, 2015 by School of Medicine Webmaster   |   Leave a Comment

Date: Friday September 25, 2015
Time: 12:00 PM until 1:00 PM
Location: MR-4 Second Floor Conference Room

Picture of eNos/GNOS vesicular trafficking pathways

Figure 1. Schematic Diagram of vesicular trafficking pathways and the location of eNOS/GSNOR interaction sites in the endothelial cell.  Proteins generated and processed in the endoplasmic reticulum (ER) are transferred to the Golgi.  In the Golgi, proteins are sorted into transport or secretory vesicles and tubular carriers for anterograde transport to the plasma membrane. Retrograde transport can occur from the plasma membrane into early then late endosomes. The transfer of proteins to the lysosome results in protein degradation.   Recycling of proteins can occur from plasma membrane to early endosome, late endosome to Golgi and Golgi to ER.   eNOS/GSNOR interaction has been detected in the Golgi and early endosome.  The functional consequences of this interaction or altered S-nitrosothiol (SNO) bioavailability as a result of this interaction in regulating intracellular transport are unknown.

SPECIFIC AIMS: Trafficking pathways in the cell ensure prompt and precise delivery of specific molecules (cargos) to different cell compartments via small vesicular and tubular carriers. Local activation of endothelial nitric oxide synthase (eNOS) generates organelle specific protein S-nitrosylation reactions that regulate intracellular transport processes.1 Pulmonary hypertension (PH) is associated with dysfunctional anterograde and retrograde vesicular transport.2  Moreover, defects in vesicular trafficking are associated with hypo-S-nitrosylation of transport proteins.3 S-nitrosoglutathione reductase (GSNOR) degrades S-nitrosoglutathione (GSNO), and via trans-nitrosation equilibrium regulates the S-nitrosothiol protein pool. Preliminary data show that eNOS and GSNOR co-immunoprecipitate in murine pulmonary endothelial cell (MPEC) lysates and that GSNO disrupts this interaction. Sucrose gradient fractionation of MPEC lysates found that this interaction is not located in the caveolin-enriched fraction (i.e. plasma membrane). Immunofluorescent labeling of eNOS and GSNOR and confocal laser scanning microscopy demonstrate that eNOS and GSNOR primarily colocalize at the cis-Golgi with lower levels seen in the trans-Golgi and endosomes. Förester resonance energy transfer (FRET) studies indicate a direct interaction between eNOS and GSNOR. Treatment of MPECs with GSNO reduces FRET efficiency, consistent with a disruption of this interaction. Interestingly, pharmacological inhibition of GSNOR with compound C3 augmented the presence of eNOS in the Golgi as well as in the plasma membranes. We hypothesize that eNOS and GSNOR colocalize to regulate local nitric oxide (NO)/S-nitrosothiol bioavailability, regulating NO signaling and intracellular trafficking. Moreover, disruption of the balance between eNOS and GSNOR may lead to endothelial dysfunction due to protein mis-location, thus contributing to the development of disease states such as PH. Defining the role of S-nitrosothiols and GSNOR in the intracellular trafficking of proteins such as eNOS (Figure 1) will be determined by three specific aims.

AIM 1 tests the hypothesis that GSNOR and eNOS colocalize in organelles involved in vesicular trafficking. Modulation of GSNOR/eNOS activities/interaction regulates intracellular transport.

AIM 2 tests the hypothesis that the S-nitrosylation status of key proteins regulating vesicular trafficking is dependent on the relative activities/interaction of eNOS/GSNOR.

AIM 3 tests the hypothesis that a mismatch in eNOS/GSNOR activities/interactions in the Golgi results in intracellular trafficking defects contributing to the gender differences seen in PH.

Overall Impact:  The turnover of the NO/S-nitrosothiol pool in endothelial cells is mediated by protein S-nitrosylation, which depends upon the interplay between eNOS and GSNOR. Taken together, our data provide compelling evidence that, (1) S-nitrosothiols and GSNOR play a vital role in intracellular trafficking of eNOS, (2) S-nitrosothiol signaling is integral to pulmonary physiology, and (3) disturbances in S-nitrosylation status directly contributes to pathology.


Contact cline@virginia.edu for more information

 

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