The Habc domain of syntaxin 3 is a ubiquitin binding domain.

Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. The syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low affinity. This domain binds efficiently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other syntaxin family members also bind to K63-linked poly-ubiquitin chains but with different chain length specificities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin may have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence. Binding of Ubiquitin to the Habc domain may regulate the function of syntaxins in membrane fusion or may suggest additional functions of this protein family.

Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells.

The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.

Soluble syntaxin 3 functions as a transcriptional regulator.

Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (syntaxin 3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.

Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes.

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral-but not the apical-plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion.

Signal transducer and activator of transcription-6 (STAT6) inhibition suppresses renal cyst growth in polycystic kidney disease.

Autosomal-dominant (AD) polycystic kidney disease (PKD) is a leading cause of renal failure in the United States, and currently lacks available treatment options to slow disease progression. Mutations in the gene coding for polycystin-1 (PC1) underlie the majority of cases but the function of PC1 has remained poorly understood. We have previously shown that PC1 regulates the transcriptional activity of signal transducer and activator of transcription-6 (STAT6). Here we show that STAT6 is aberrantly activated in cyst-lining cells in PKD mouse models. Activation of the STAT6 pathway leads to a positive feedback loop involving auto/paracrine signaling by IL13 and the IL4/13 receptor. The presence of IL13 in cyst fluid and the overexpression of IL4/13 receptor chains suggests a mechanism of sustained STAT6 activation in cysts. Genetic inactivation of STAT6 in a PKD mouse model leads to significant inhibition of proliferation and cyst growth and preservation of renal function. We show that the active metabolite of leflunomide, a drug approved for treatment of arthritis, inhibits STAT6 in renal epithelial cells. Treatment of PKD mice with this drug leads to amelioration of the renal cystic disease similar to genetic STAT6 inactivation. These results suggest STAT6 as a promising drug target for treatment of ADPKD.

Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment.

PURPOSE: We previously reported that autologous dendritic cells pulsed with acid-eluted tumor peptides can stimulate T cell-mediated antitumor immune responses against brain tumors in animal models. As a next step in vaccine development, a phase I clinical trial was established to evaluate this strategy for its feasibility, safety, and induction of systemic and intracranial T-cell responses in patients with glioblastoma multiforme. EXPERIMENTAL DESIGN: Twelve patients were enrolled into a multicohort dose-escalation study and treated with 1, 5, or 10 million autologous dendritic cells pulsed with constant amounts (100 mug per injection) of acid-eluted autologous tumor peptides. All patients had histologically proven glioblastoma multiforme. Three biweekly intradermal vaccinations were given; and patients were monitored for adverse events, survival, and immune responses. The follow-up period for this trial was almost 5 years. RESULTS: Dendritic cell vaccinations were not associated with any evidence of dose-limiting toxicity or serious adverse effects. One patient had an objective clinical response documented by magnetic resonance imaging. Six patients developed measurable systemic antitumor CTL responses. However, the induction of systemic effector cells did not necessarily translate into objective clinical responses or increased survival, particularly for patients with actively progressing tumors and/or those with tumors expressing high levels of transforming growth factor beta(2) (TGF-beta(2)). Increased intratumoral infiltration by cytotoxic T cells was detected in four of eight patients who underwent reoperation after vaccination. The magnitude of the T-cell infiltration was inversely correlated with TGF-beta(2) expression within the tumors and positively correlated with clinical survival (P = 0.047). CONCLUSIONS: Together, our results suggest that the absence of bulky, actively progressing tumor, coupled with low TGF-beta(2) expression, may identify a subgroup of glioma patients to target as potential responders in future clinical investigations of dendritic cell-based vaccines.

Modulation of major histocompatibility complex Class I molecules and major histocompatibility complex-bound immunogenic peptides induced by interferon-alpha and interferon-gamma treatment of human glioblastoma multiforme.

OBJECT: Little is known about the quantitative modulation of major histocompatibility complex (MHC) Class I expression on human gliomas that is effected by interferons; even less is known about the immunogenic peptides that are accommodated in the peptide-binding motifs of MHC Class I alleles in these brain tumors. In this article the authors investigated the ability of interferon (IFN)alpha and IFNgamma to upregulate MHC Class I expression and to modulate acid-eluted Class I-bound peptides on human glioblastoma multiforme (GBM) cells in vitro. METHODS: Early-passage primary human GBM cell cultures and U87MG GBM cells were incubated with varying doses of INFalpha or IFNgamma ranging between 0 and 2000 U/ml. Upregulation of MHC Class I expression was assayed by immunocytochemical analysis, flow cytometry, and Western blot analysis. Modulation of acid-eluted MHC Class I-bound peptides from the IFN-treated GBM cells was examined with the aid of mass spectroscopy. The in vitro expression of the MHC Class I molecule was upregulated by both IFNalpha and IFNgamma in a dose-dependent manner. Interferon-gamma exhibited a more potent effect on MHC Class I upregulation, peaking at 10 U/ml; whereas the effect of IFNalpha was less marked, reaching a plateau at 500 U/ml. In addition, a native peptide eluted from MHC Class I molecules of human GBM cells was identified and found to be consistently upregulated by IFN treatment. CONCLUSIONS: Interferon-alpha and IFN-gamma can significantly upregulate the MHC Class I molecules that are expressed on the cell surface of human GBM cells as well as the potentially immunogenic peptides bound to the MHC. These results may help explain the molecular basis for increased immunogenicity with IFN treatment of human GBMs and might provide added insight into the design of future antitumor vaccines for human brain tumors.