MMP-9 Processing of Intestinal Smooth Muscle-derived GDNF is Required for Neurotrophic Action on Enteric Neurons.

The neurotrophin GDNF guides development of the enteric nervous system (ENS) in embryogenesis and directs survival and axon outgrowth in postnatal myenteric neurons in vitro. GDNF expression in intestinal smooth muscle cells is dynamic, with upregulation by inflammatory cytokines in vitro or intestinal inflammation in vivo, but the role of post-translational proteolytic cleavage is undefined. In a co-culture model of myenteric neurons, smooth muscle and glia, inhibition of serine or cysteine protease activity was ineffective against the >2-fold increase in axon density caused by TNFα. However, inhibitors of metalloproteinases (MMP) identified an essential role of MMP-9, and qPCR and western blotting showed that pro-inflammatory cytokines increased both mRNA and protein expression for MMP-9, in both cellular lysates and conditioned medium (CM). Inhibition of MMP-9 prevented the cytokine-induced increase in mature GDNF in CM or cellular lysates of co-cultures or cell lines of intestinal smooth muscle cells (ISMC) from adult rat colon. Western blotting showed parallel upregulation of mature GDNF and MMP-9 vs control in ISMC isolated on Day 2 of TNBS-induced colitis. Nonetheless, transfection of GDNF plasmid into HEK-293 cells as a carrier system, or directly into the co-culture model, conveyed a strong neurotrophic effect that was MMP-9 dependent. We conclude that MMP-9 activity is required for the neurotrophic effects of GDNF on myenteric neurons in vitro. However, the coordinated upregulation of GDNF and MMP-9 in intestinal smooth muscle by inflammatory cytokines provides a supportive, target cell-derived environment that limits inflammatory damage to the ENS.

Cholesterol is required for efficient endoplasmic reticulum-to-Golgi transport of secretory membrane proteins.

Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3-6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss of mobility of proteins in the ER lumen. This impaired lateral mobility is correlated with impaired ER-to-Golgi transport. These results provide evidence for the first time that cholesterol is required in the ER membrane to maintain mobility of membrane proteins and thus protein secretion.

Purification and functional properties of prenylated Rab acceptor 2.

PRA2 was found to interact with the ER-localized protein VAMP-associated protein of 33 kDa or VAP-33 by a yeast two-hybrid screen. We describe here the purification of PRA2 and VAP-33 as well as an in vitro pull-down procedure to verify the interaction. PRA2 was found to form a large sodium dodecyl sulfate (SDS)-insoluble complex upon heat denaturation, resulting in significant reduction in the Western immunoblot signal. This phenomenon is specific to PRA2 and was not observed with PRA1. We also found that protein interaction with PRA2 is highly sensitive to detergent and describe a covalent cross-linking procedure for mammalian cell extracts to stabilize the PRA2-containing complex prior to membrane solubilization and immunoprecipitation.

FFAT rescues VAPA-mediated inhibition of ER-to-Golgi transport and VAPB-mediated ER aggregation.

The VAMP-associated proteins termed VAP are a small gene family of proteins characterised by the presence of an N-terminal major sperm protein (MSP) domain. The P56S mutation of the B isoform (VAPB) has been linked to late-onset amyotrophic lateral sclerosis (ALS8) and its expression causes formation of large ER aggregates. Overexpression of the wild-type A isoform (VAPA) but not the B isoform (VAPB), inhibited ER-to-Golgi transport of membrane proteins. This transport block by VAPA was primarily due to decreased segregation of membrane cargo into ER vesicles. We also found that VAPA inhibited lateral diffusion of membrane proteins, most likely through its stable association with microtubules. The MSP domain of VAP is known to interact with the FFAT motif (two phenylalanines in an acidic tract) of proteins involved in sterol regulation. Overexpression of FFAT restored ER-to-Golgi transport and lateral diffusion of membrane proteins, and resolved the large ER aggregates in VAPB-P56S. Application of a FFAT peptide restored in vitro ER vesicle budding and disrupted VAP-microtubule association. Thus, overexpression of the two VAP isoforms causes retention of ER membrane proteins by impeding lateral diffusion and their incorporation into transport vesicles. This inhibitory effect can be relieved by expression of the FFAT motif.

Disruption of Golgi morphology and trafficking in cells expressing mutant prenylated rab acceptor-1.

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization. Class A mutants were retained in the endoplasmic reticulum (ER) and exerted an inhibitory effect on transport of vesicular stomatitis virus envelope glycoprotein (VSVG) from the ER to Golgi as well as to the plasma membrane. Class B mutants exhibited a highly condensed Golgi complex and inhibited exit of anterograde cargo from this organelle. Class C mutants exhibited an intermediate phenotype with Golgi and ER localization along with extensive tubular structures emanating from the Golgi complex. There was a direct correlation between the cellular phenotype and binding to Rab and VAMP2. Class A and C mutants showed a significant decrease in Rab and VAMP2 binding, whereas an increase in binding was observed in the class B mutants. Thus, PRA1 is required for vesicle formation from the Golgi complex and might be involved in recruitment of Rab effectors and SNARE proteins during cargo sequestration.