The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi.

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of .

Modeling the dynamic behaviors of the COPI vesicle formation regulators, the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes.

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat.

Enterovirus Infection Induces Massive Recruitment of All Isoforms of Small Cellular Arf GTPases to the Replication Organelles.

Enterovirus replication requires the cellular protein GBF1, a guanine nucleotide exchange factor for small Arf GTPases. When activated, Arfs associate with membranes, where they regulate numerous steps of membrane homeostasis. The requirement for GBF1 implies that Arfs are important for replication, but which of the different Arfs function(s) during replication remains poorly understood.

Wnt signaling modulator DKK1 as an immunotherapeutic target in ovarian cancer.

Objectives: Wnt pathway mutations are a hallmark of endometrioid and clear cell subtypes of epithelial ovarian carcinoma (EOC). However, no drugs targeting the Wnt pathway in EOC are FDA-approved. Dickkopf-related protein 1 (DKK1), a modulator of the Wnt pathway, has emerged as a promising therapeutic target.

Biomarkers in ovarian cancer: To be or not to be.

Biomarkers are becoming increasingly important in the treatment of epithelial ovarian cancer. Recent work from many laboratories has begun to provide clinically meaningful biomarkers. This review summarizes the state of the science regarding biomarkers for stratifying early-stage patients into those who benefit from adjuvant treatment, primary debulking versus interval debulking, and specific targeted therapy.

Benzyloxycarbonyl-proline-prolinal (ZPP): Dual complementary roles for neutrophil inhibition.

Neutrophil influx and activation contributes to organ damage in several major lung diseases. This inflammatory influx is initiated and propagated by both classical chemokines such as interleukin-8 and by downstream mediators such as the collagen fragment cum neutrophil chemokine Pro-Gly-Pro (PGP), which share use of the ELR + CXC receptor family. Benzyloxycarbonyl-proline-prolinal (ZPP) is known to suppress the PGP pathway via inhibition of prolyl endopeptidase (PE), the terminal enzyme in the generation of PGP from collagen.

Proline-Glycine-Proline Peptides Are Critical in the Development of Smoke-induced Emphysema.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide and is characterized by an excessive airway neutrophilic response. The neutrophil chemoattractant proline-glycine-proline (PGP) and its more potent acetylated form (acPGP) have been found to be elevated in patients with COPD and act via CXCR2. Here, we investigated the impact of neutralizing PGP peptides in a murine model for emphysema.

Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung.

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome.

Exosomal microRNA predicts and protects against severe bronchopulmonary dysplasia in extremely premature infants.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls.

MicroRNA-145 Antagonism Reverses TGF-β Inhibition of F508del CFTR Correction in Airway Epithelia.

Rationale: MicroRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of particular interest in cystic fibrosis (CF) as it has a direct binding site in the 3'-untranslated region of CFTR (cystic fibrosis transmembrane conductance regulator) and is upregulated by the CF genetic modifier TGF (transforming growth factor)-β.

Objectives: To demonstrate that miR-145 mediates TGF-β inhibition of CFTR synthesis and function in airway epithelia.

Genetic regulation of expression of leukotriene A4 hydrolase.

In chronic inflammatory lung disorders such as chronic obstructive pulmonary disease (COPD), the concurrent organ-specific and systemic inflammatory responses lead to airway remodelling and vascular dysfunction. Although a major common risk factor for COPD, cigarette smoke alone cannot explain the progression of this disease; there is increasing evidence that genetic predisposition also plays a role in COPD susceptibility and progression. A key enzyme in chronic lung inflammation is leukotriene A4 hydrolase (LTA4H).

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown.

Alterations in blood leukocytes of G551D-bearing cystic fibrosis patients undergoing treatment with ivacaftor.

Background: Ivacaftor improves clinical outcome by potentiation of mutant G551D CFTR. Due to the presence of CFTR in monocytes and polymorphonuclear neutrophils (PMNs), we hypothesized that ivacaftor may impact leukocyte activation.

Methods: We examined blood leukocytes from G551D CF subjects prior to and at one and six months after receiving ivacaftor.

An aberrant leukotriene A4 hydrolase-proline-glycine-proline pathway in the pathogenesis of chronic obstructive pulmonary disease.

Rationale: Chronic neutrophilic inflammation is a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD) and persists after cigarette smoking has stopped. Mechanisms involved in this ongoing inflammatory response have not been delineated.

Objectives: We investigated changes to the leukotriene A4 hydrolase (LTA4H)-proline-glycine-proline (PGP) pathway and chronic inflammation in the development of COPD.

Pulmonary matrix metalloproteinase-9 activity in mechanically ventilated children with respiratory syncytial virus.

Respiratory syncytial virus (RSV) infection is a potent stimulus for airway epithelial expression of matrix metalloproteinase (MMP)-9. MMP-9 activity in vivo is a predictor of disease severity in children with RSV-induced respiratory failure. Human airway epithelial cells were infected with RSV A2 strain and analysed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 (a natural inhibitor of MMP-9) release.

The Sec7 guanine nucleotide exchange factor GBF1 regulates membrane recruitment of BIG1 and BIG2 guanine nucleotide exchange factors to the trans-Golgi network (TGN).

Three Sec7 guanine nucleotide exchange factors (GEFs) activate ADP-ribosylation factors (ARFs) to facilitate coating of transport vesicles within the secretory and endosomal pathways. GBF1 recruits COPI to pre-Golgi and Golgi compartments, whereas BIG1 and BIG2 recruit AP1 and GGA clathrin adaptors to the trans-Golgi network (TGN) and endosomes. Here, we report a functional cascade between these GEFs by showing that GBF1-activated ARFs (ARF4 and ARF5, but not ARF3) facilitate BIG1 and BIG2 recruitment to the TGN.

Identification of a functional domain within the p115 tethering factor that is required for Golgi ribbon assembly and membrane trafficking.

The tethering factor p115 (known as Uso1p in yeast) has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that depletion of p115 by using RNA interference (RNAi) in C.

COPII and COPI traffic at the ER-Golgi interface.

Protein traffic is necessary to maintain homeostasis in all eukaryotic organisms. All newly synthesized secretory proteins destined to the secretory and endolysosmal systems are transported from the endoplasmic reticulum to the Golgi before delivery to their final destinations. Here, we describe the COPII and COPI coating machineries that generate carrier vesicles and the tethers and SNAREs that mediate COPII and COPI vesicle fusion at the ER-Golgi interface.

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation.

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ∼200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d.

The Garz Sec7 domain guanine nucleotide exchange factor for Arf regulates salivary gland development in Drosophila.

Surface delivery of proteins involved in cell-cell and cell-matrix interactions in cultured mammalian cells requires the GBF1 guanine nucleotide exchange factor. However, the role of GBF1 in delivery of adhesion proteins during organogenesis in intact animals has not been characterized. Here, we report the function of the fly GBF1 homolog, Gartenzwerg (Garz) in the development of the salivary gland in Drosophila melanogaster.

Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi biogenesis and protein trafficking.

COPI recruitment to membranes appears to be essential for the biogenesis of the Golgi and for secretory trafficking. Preventing COPI recruitment by expressing inactive forms of the ADP-ribosylation factor (ARF) or the ARF-activating guanine nucleotide exchange factor GBF1, or by treating cells with brefeldin A (BFA), causes the collapse of the Golgi into the endoplasmic reticulum (ER) and arrests trafficking of soluble and transmembrane proteins at the ER. Here, we assess COPI function in Golgi biogenesis and protein trafficking by preventing COPI recruitment to membranes by removing GBF1.

Activation of ADP-ribosylation factor regulates biogenesis of the ATP7A-containing trans-Golgi network compartment and its Cu-induced trafficking.

ATP7A (MNK) regulates copper homeostasis by translocating from a compartment localized within the trans-Golgi network to the plasma membrane (PM) in response to increased copper load. The mechanisms that regulate the biogenesis of the MNK compartment and the trafficking of MNK are unclear. Here we show that the architecture of the MNK compartment is linked to the structure of the Golgi ribbon.

On and off membrane dynamics of the endoplasmic reticulum-golgi tethering factor p115 in vivo.

The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 approximately 20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 approximately 13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP.