Stuart A. Ross

Characterization of the Insulin-regulated Membrane Aminopeptidase in 3T3-L1 Adipocytes

A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.

Crystallographic Studies of Human MitoNEET

MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33–108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster.

Protective properties afforded by pioglitazone against intrastriatal LPS in Sprague–Dawley rats

We created an inflammation-induced Parkinsons disease model, where microglia activation leads to oxidative stress, mitochondrial dysfunction, and dopaminergic neurodegeneration in the substantia nigra. Pioglitazone, an agonist of peroxisome proliferator activated receptor-gamma (PPAR-gamma), can prevent these deficits and protect dopaminergic neurons. To continue exploring the effects of pioglitazone in this model we focused on the expression of PPAR-gamma, uncoupling protein 2 (UCP2), and mitoNEET. We report that intrastriatal lipopolysaccharide (LPS) increases striatal PPAR-gamma, UCP2, and mitoNEET expression, and pioglitazone attenuates these LPS-induced changes.

Cloning and characterization of a 22 kDa protein from rat adipocytes: a new member of the reticulon family

In the course of our examination of proteins associated with the GLUT4-containing vesicles of rat adipocytes we have identified a new 22 kDa member of the family of endoplasmic reticulum (ER) proteins known as reticulons. The protein, which we refer to as vp20, was purified from a preparation of GLUT4-containing vesicles of rat adipocytes, and tryptic peptides were micro-sequenced. From this information a cDNA encoding a single open reading frame for a protein of 22 kDa was cloned. This protein is homologous to known members of the reticulon protein family. vp20 has two hydrophobic stretches of about 35 amino acids that could be membrane spanning domains and an ER retention motif at its carboxy-terminus. vp20 was most abundant in the high density microsome fraction of adipocytes, which is the fraction most enriched in ER. Only a small fraction of vp20 was present in the GLUT4 vesicle population, and that fraction appears to be due to ER vesicles that were non-specifically bound to the adsorbent. Analysis of tissue distribution of vp20 in rats revealed that it is concentrated in muscle, fat and the brain.

Cloning and preliminary characterization of a 121 kDa protein with multiple predicted C2 domains.

In the course of characterizing proteins present in a preparation of vesicles from rat adipocytes containing glucose transporters, we examined a protein that migrated at 115 kDa upon SDS gel electrophoresis (designated vp115). Sequences of tryptic peptides were obtained, and from this information the cDNA for rat vp115 was cloned. The cDNA encodes an open reading frame for a protein of 121 kDa. Computer-aided sequence analysis predicted that vp115 has a potential membrane-inserted or membrane-spanning domain toward its amino terminus, followed by five C2 domains. Immunoblotting revealed that vp115 was not actually a component of the glucose transporter-containing vesicles, was most abundant in the plasma membranes and high density microsome fractions of rat adipocytes, and was expressed in all the major rat tissues.

The Pharmacology of Caveolae

Based on work in the last 15 years, caveolae are now recognized as rather complex and dynamic plasma membrane domains with important roles in cellular uptake of molecules, signal transduction, lipid homeostasis, and tumorigenesis. Functionally, caveolae orchestrate very specific events in distinct cell types, thereby making these organelles one of the most interesting compartments in cells. In this chapter, we will discuss the salient features of these active cellular domains. We begin with a general introduction to the biochemistry and cell biology of caveolae, describe some of the myriad signaling events that initiate in caveolae (or traffic through caveolae) and how they can go awry in cancer, describe how caveolar proteins can be exploited for novel drug discovery, and finally provide commentary over a growing body of evidence that suggests how molecules are presented to caveolae makes a difference in the pharmacological effects that ensue.