Inflammatory bowel disease (IBD) is a chronic complex inflammatory gut pathological condition, examples of which include Crohn’s disease (CD) and ulcerative colitis (UC), which is associated with significant morbidity. Although the etiology of IBD is unknown, gut microbiota alteration (dysbiosis) is considered a novel factor involved in the pathogenesis of IBD. The gut microbiota acts as a metabolic organ and contributes to human health by performing various physiological functions; deviation in the gut flora composition is involved in various disease pathologies, including IBD. This review aims to summarize the current knowledge of gut microbiota alteration in IBD and how this contributes to intestinal inflammation, as well as explore the potential role of gut microbiota-based treatment approaches for the prevention and treatment of IBD. The current literature has clearly demonstrated a perturbation of the gut microbiota in IBD patients and mice colitis models, but a clear causal link of cause and effect has not yet been presented. In addition, gut microbiota-based therapeutic approaches have also shown good evidence of their effects in the amelioration of colitis in animal models (mice) and IBD patients, which indicates that gut flora might be a new promising therapeutic target for the treatment of IBD. However, insufficient data and confusing results from previous studies have led to a failure to define a core microbiome associated with IBD and the hidden mechanism of pathogenesis, which suggests that well-designed randomized control trials and mouse models are required for further research. In addition, a better understanding of this ecosystem will also determine the role of prebiotics and probiotics as therapeutic agents in the management of IBD.

The ADP-ribosylation factors (Arfs) are six proteins within the larger Arf family and Ras superfamily that regulate membrane traffic. Arfs all share numerous biochemical activities and have very similar specific activities. The use of dominant mutants and brefeldin A has been important to the discovery of the cellular functions of Arfs but lack specificity between Arf isoforms. We developed small interference RNA constructs capable of specific depletion of each of the cytoplasmic human Arfs to examine the specificity of Arfs in live cells. No single Arf was required for any step of membrane traffic examined in HeLa cells. However, every combination of the double knockdowns of Arf1, Arf3, Arf4, and Arf5 yielded a distinct pattern of defects in secretory and endocytic traffic, demonstrating clear specificity for Arfs at multiple steps. These results suggest that the cooperation of two Arfs at the same site may be a general feature of Arf signaling and provide candidates at several cellular locations that when paired with data on the localization of the many different Arf guanine nucleotide exchange factors, Arf GTPase activating proteins, and effectors will aid in the description of the mechanisms of specificity in this highly conserved and primordial family of regulatory GTPases.

A family of three structurally related proteins were cloned from human cDNA libraries by their ability to interact preferentially with the activated form of human ADP-ribosylation factor 3 (ARF3) in two-hybrid assays. The specific and GTP-dependent binding was later confirmed through direct protein binding of recombinant proteins. The three proteins share large (Ϸ300 residues) domains at their N termini that are 60 -70% identical to each other and a shorter (73 residues) domain at their C termini with 70% homology to the C-terminal "ear" domain of ␥-adaptin. Although GGA1 is found predominantly as a soluble protein by cell fractionation, all three proteins were found to localize to the trans-Golgi network (TGN) by indirect immunofluorescence. The binding of GGAs to TGN was sensitive to brefeldin A, consistent with this being an ARF-dependent event. Thus, these proteins have been named Golgi-localizing, ␥-adaptin ear homology domain, ARF-binding proteins, or GGAs. The finding that overexpression of GGAs was sufficient to alter the distribution of markers of the TGN (TGN38 and mannose 6-phosphate receptors) led us to propose that GGAs are effectors for ARFs that function in the regulation of membrane traffic through the TGN.

Subcellular distributions of the five human Arf proteins were examined, using a set of isoform-specific polyclonal and a pan-Arf monoclonal antibodies. Subcellular fractionation of cultured mammalian cells allowed the demonstration that Arf6 is uniquely localized to the plasma membranes of Chinese hamster ovary cells. The plasma membrane distrubution was unaffected by either GTP␥S (guanosine 5-O-(3-thio)triphosphate) or brefeldin A, an activator and inhibitor of Arf activities, respectively. In contrast, Arf proteins 1, 3, 4, and 5 were predominantly cytosolic but could be recruited to a variety of intracellular membranes, but not plasma membranes, upon incubation in the presence of GTP␥S. The GTP␥S-promoted binding of the cytosolic Arf proteins to membranes was blocked by brefeldin A. The stable association of Arf6 with plasma membranes and the insensitivity of its localization to either GTP␥S or brefeldin A revealed a clear distinction between Arf6 and the other Arf isoforms. Localization of Arf6 to the plasma membrane suggests a unique cellular role for this isoform at the plasma membrane, but failure to find endogenous Arf6 on endocytic structures, including clathrin-coated vesicles, appears inconsistent with the proposed role of Arf6 in assembly of coat structures or endosomes in transfected fibroblasts (1, 2).The ADP-ribosylation factor (Arf) 1 family is a group of structurally related proteins that form a subset of the Ras superfamily of regulatory GTP-binding proteins (for a recent review, see Ref. 3). In addition to serving as cofactors for cholera toxin-catalyzed ADP-ribosylation, Arf proteins have more recently been associated with a wide array of functions. These include acting as regulators of the binding of coat proteins and adaptins to intracellular membranes (4, 5), activators of phospholipase D (6, 7), regulators of ER and Golgi morphology and function (8, 9), and cytosolic factors conferring sensitivity to GTP␥S in cell-free assays of intra-Golgi (10 -12) and ER-Golgi transport (13), and endosome (14) and nuclear membrane fusion (15, 16).The importance of Arf proteins in both membrane traffic and organelle organization was manifest due to the sensitivities of most Arf proteins to both GTP␥S, a slowly hydrolyzable GTP analog, and to brefeldin A (BFA), a fungal metabolite capable of inhibiting guanine nucleotide exchange on Arf in a crude system (17-19). The activation (GTP binding) and deactivation (GTP hydrolysis) cycle of Arf action in cells is thought to coincide with its binding and release, respectively, from intracellular membranes. In this model, activation of a soluble Arf protein results in its translocation to a membrane and the recruitment, through unknown mechanisms, of coat proteins or adaptor complexes to that membrane. It remains unclear how, or even if, Arf-mediated activation of phospholipase D or cholera toxin relate to mechanisms of regulation of membrane transport by Arf proteins.

Abstract. ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mxl promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and B-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed,

In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and ␤-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and ␤-COP, expression of a GTPase-deficient form of ARF (ARF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (Teal et al., 1994; Zhang et al., 1994). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.

Arf proteins are ubiquitous, eukaryotic regulators of virtually every step of vesicular membrane traffic. ADPribosylation factors are essential in yeast and the lethality resulting from either overexpression or underexpression (deletion) of Arf genes has previously been ascribed to dysregulation of the secretory process. We have identified a family of four genes (Suppressors of Arf t s , SAT) as high copy suppressors of a loss of function allele of ARF1 (arf1-3). Those proteins with SAT activity were found to contain a minimal consensus motif, including a C2C2H2 cluster with a novel and specific spacing. Genetic interactions between members of this family and with ARF1 are consistent with each sharing a common cellular pathway. Included in this family is Gcs1, a protein previously described (Poon, P. P., Wang, X., Rotman, M., Huber, I., Cukierman, E., Cassel, D., Singer, R. A., and Johnston, G. C. Suppression of the growth defect of arf1 ؊3 cells was observed under conditions that did not alter the secretory defect associated with arf1 ؊ mutation, indicating that the essential role of Arf in eukaryotes can be distinguished from role(s) in the secretory pathway and appear to employ distinct pathways and effectors. ADP-ribosylation factors (Arfs)1 are the family of monomeric, 21-kDa GTP-binding proteins originally identified as protein co-factors for cholera toxin-catalyzed ADP-ribosylation of G s , the heterotrimeric G protein activator of adenylyl cyclase (1, 2). Studies have implicated Arfs as regulators of a number of steps of vesicular membrane transport, coat protein assembly, and maintenance of the integrity of the ER and Golgi compartments Either overexpression or deletion of (both) ARF genes is lethal to yeast cells (9). The two yeast Arf proteins are 96% identical, and no phenotype has been defined for the loss of ARF2. In contrast, arf1 Ϫ cells grow slower than parental controls at all temperatures are weakly cold-sensitive (c s ), defective in the ability to process secreted proteins, e.g. invertase, and are supersensitive to fluoride (9, 10). Supersensitivity to fluoride results from an unknown mechanism, but it has proven a useful and specific indicator of loss of Arf1 function. A second copy of ARF2 complements these phenotypes associated with arf1 Ϫ cells. The 5-10-fold higher level of expression of Arf1 over Arf2 is the likely explanation for the differences in phenotypes between arf1 Ϫ and arf2 Ϫ (11). For this reason, we focused our efforts on genetic studies of ARF1, and the studies described below were conducted in an arf2 Ϫ background, unless otherwise indicated. MATERIALS AND METHODS Yeast CultureYeast were cultured using standard conditions, as described in Sherman et al. (12). Selective plates containing fluoride were prepared according to Stearns et al. (11). Yeast transformations were performed by the PEG/LiCl method of Schiestl et al. (13). MutagenesisPCR Mutagenesis-Random mutagenesis of the open reading frame of the ARF1 gene was achieved by PCR under conditions of reduced stringency;...

Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly2 or Asp26 resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp26 (homologous to Gly12 of p21ras) was essential for the binding of the activating nucleotide, guanosine 5'-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21ras or Gs alpha, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins. Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have approximately 300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.