Alan G. Wildeman

V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway.

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.

Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages

After internalization into macrophages non‐pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago‐lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill ‘non‐pathogenic’Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V‐ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.

Deposition of genetically engineered human antibodies into the egg yolk of hens

To determine if human immunoglobulins (hIg) are capable of being transported into the hens egg, 10 microg each of purified hIgG and hIgA were intravenously injected into SC Hyline(TM) hens and their presence in egg yolk and egg white was determined by ELISA. In both cases deposition into the egg yolk was observed and in the case of hIgA, deposition was also observed in the egg white. Two stably transfected DT40 cell lines secreting recombinant human IgG3 and IgA (rhIgG3 and rhIgA) were injected into laying hens. The DT40 cells colonized the host and rhIgG3 and rhIgA were deposited in egg yolk. Deposition of rhIgA was also observed in the egg white. These data demonstrate that human immunoglobulins and other foreign proteins may be targeted to the chickens egg. In view of the high rate of reproduction, the short generation interval, the high rates of egg production and the extensive infrastructure to fractionate egg yolk proteins, it should be possible to produce large amounts of foreign protein in the eggs of transgenic chickens.

β1 Integrin Binds the 16-kDa Subunit of Vacuolar H+-ATPase at a Site Important for Human Papillomavirus E5 and Platelet-derived Growth Factor Signaling

Integrins mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction. We have identified an interaction between the β1 integrin and the 16-kDa subunit of vacuolar H+-ATPase (16K). This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and inin vitro binding assays using bacterially expressed proteins. Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged 16K demonstrate co-localization with β1 integrin in focal adhesions. Deletion of the fourth of four transmembrane helices in 16K results in loss of interaction with β1 integrin in vitroand in the two-hybrid system, and less prominent staining in focal adhesions. This helix is also required for ligand-independent activation of platelet-derived growth factor-β receptor signaling by the human papillomavirus E5 oncoprotein. Overexpression of 16K or expression of 16K lacking this helix alters the morphology of myoblasts and fibroblasts, suggesting that the interaction of 16K with integrins could be important for cell growth control. We also discuss the possible role 16K might play in integrin movement.

Stage-dependent Redistribution of the V-ATPase During Bovine Implantation

The 16-kD subunit of the vacuolar H+ -ATPase (V-ATPase), or ductin, is essential for the activity of this proton pump and has roles in intercellular communication and control of cell growth and differentiation. The V-ATPase is important for acidification-dependent degradation of tissue matrices through which some cell types move, and for pH regulation across some epithelial cell layers. Placentation involves intricate signaling, cell proliferation, and controlled invasion. We examined the distribution of three subunits of the V-ATPase in bovine trophoblast and endometrium at the time of implantation to determine the relationship of ductin expression to that of two other subunits, A (approximately 73 kD) and B (approximately 58 kD). Epithelial expression of all three subunits was observed, and in nonpregnant animals this expression was apical. As pregnancy proceeded, expression of all subunits became pericellular in luminal but not glandular epithelium, suggesting a redistribution of V-ATPase activity. The trophoblast expressed all three subunits during initial contact with the epithelium. In the stroma, ductin expression was reduced after implantation, and we discuss the possibility that ductin plays a role in the shifting communication between stromal and epithelial cells induced by embryo attachment.

Oligomerization of the E5 protein of human papillomavirus type 16 occurs through multiple hydrophobic regions.

The high risk forms of human papillomavirus (HPV) (primarily types 16 and 18) are the leading cause of cervical cancer worldwide. Infection results in expression of three oncoproteins, E5, E6, and E7, the latter two being of predominant importance in maintaining a transformed state of the host epithelial cell. While little is known about the role(s) of the HPV E5, the bovine papillomavirus type 1 (BPV1) E5 protein has been well characterized. A study of HPV16 E5 was performed, focusing on the proteins ability to self-interact, its ability to bind to the 16-kDa subunit of the vacuolar H(+)-ATPase (16K), and its cellular localization. As has been previously shown for BPV1 E5, we found that HPV16 E5 is also capable of self-interaction and binding to 16K. Further, we examined which portions of the HPV16 E5 protein were involved in these interactions using progressive deletions of putative transmembrane helices of the protein. All of the E5 deletion mutants tested bound to full-length E5 as well as to 16K, suggesting that these protein-protein interactions are based on hydrophobic interactions. The majority of E5 expressed in HEK 293-T7 cells was perinuclear but did not appear to localize to the cis/medial-Golgi, in contrast to previous reports for both HPV16 E5 and BPV1 E5.

Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.

Suppression of Tumor-related Glycosylation of Cell Surface Receptors by the 16-kDa Membrane Subunit of Vacuolar H+-ATPase

The glycosylation of integrins and other cell surface receptors is altered in many transformed cells. Notably, an increase in the number of β1,6-branched N-linked oligosaccharides correlates strongly with invasive growth of cells. An ectopic expression of the Golgi enzymeN-acetylglucosaminyltransferase V (GlcNAc-TV), which forms β1,6 linkages, promotes metastasis of a number of cell types. It is shown here that the 16-kDa transmembrane subunit (16K) of vacuolar H+-ATPase suppresses β1,6 branching of β1integrin and the epidermal growth factor receptor. Overexpression of 16K inhibits cell adhesion and invasion. 16K contains four hydrophobic membrane-spanning α-helices, and its ability to influence glycosylation is localized primarily within the second and fourth membrane-spanning α-helices. 16K also interacts directly with the transmembrane domain of β1 integrin, but its effects on glycosylation were independent of its binding to β1integrin. These data link cell surface tumor-related glycosylation to a component of the enzyme responsible for acidification of the exocytic pathway.

A novel strategy for regulated expression of a cytotoxic gene.

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.

Studies on the use of plant extracts in assessing the effects of plant metabolism on the mutagenicity and toxicity of pesticides

We have carried out studies on the effects of plant metabolism on the mutagenicity of agricultural chemicals. Our approach is to use a cell-free plant extract, as a source of metabolic enzymes, in a standard Ames test. Using a number of test compounds, we observe that plant metabolism can alter the mutagenicity of several pesticides, and can in some instances give rise to metabolites apparently unique from those which are formed in animal cells. A number of parameters of the assay have been examined, and we find that the assay temperature and preincubation of the pesticide with the extract can significantly alter the outcome of the test. We also have devised a method of controlling for the effects that natural extracts can have on the spontaneous reversion rate of the Salmonella tester strains, in an effort to distinguish slight mutagenic responses from the effects of nutrients (e.g. histidine for his- bacteria) in the assay.