scholarly journals GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors

2002 ◽  
Vol 365 (2) ◽  
pp. 369-378 ◽  
Author(s):  
Hiroyuki TAKATSU ◽  
Kaori YOSHINO ◽  
Kyoko TODA ◽  
Kazuhisa NAKAYAMA
Keyword(s):  
Membrane Trafficking ◽  
Class Ii ◽  
Small Gtpases ◽  
Class I ◽  
Class Iii ◽  
Limited Region ◽  
Adp Ribosylation ◽  
Early Endosomes

ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1—ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1—ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1—GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, γ-adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.

The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3116-3116
Author(s):  
Aleksandra Franovic ◽  
Nichol Miller ◽  
Paul Severson ◽  
Toufike Kanouni ◽  
Noelito Timple ◽  
...  
Keyword(s):  
Class Ii ◽  
Class I ◽  
Braf Inhibitors ◽  
Class Iii ◽  
Braf Gene ◽  
Braf Mutations ◽  
Braf Mutant ◽  
Gene Alterations

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 < 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

scholarly journals Uropathogenic Escherichia coli Flagella Aid in Efficient Urinary Tract Colonization

2005 ◽  
Vol 73 (11) ◽  
pp. 7657-7668 ◽  
Author(s):  
Kelly J. Wright ◽  
Patrick C. Seed ◽  
Scott J. Hultgren
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Class I ◽  
Class Iii ◽  
Epithelial Surface ◽  
Fitness Advantage ◽  
Regulatory Cascade ◽  
Tract Infections

ABSTRACT In the murine model of urinary tract infections (UTI), cystitis by uropathogenic Escherichia coli (UPEC) occurs through an intimate relationship with the bladder superficial umbrella cell entailing cycles of adherence, invasion, intracellular bacterial community (IBC) formation, and dispersal (fluxing) from the intracellular environment. IBC dispersal is a key step that results in the spread of bacteria over the epithelial surface to initiate additional rounds of IBC formation. We investigated the role of flagella in mediating adherence and motility during UTI, hypothesizing that the dispersion of the IBC would be incomplete in the absence of motility, thus interrupting the IBC pathway and attenuating the infection. Using gfp reporter fusions, the expression of the flagellar class I flhDC and class III fliC genes was monitored to track key points of regulation throughout the pathogenic cascade. In vitro, growth under conditions promoting motility resulted in the robust expression of both fusions. In contrast, only the class I fusion produced significant expression throughout early stages of IBC development including the dispersion stage. Thus, unlike in vitro modeling of motility, the regulatory cascade appeared incomplete in vivo. Throughout IBC formation, nonmotile ΔfliC mutants achieved the same number of IBCs as the wild-type (wt) strain, demonstrating that flagella are neither essential nor required for first- or second-generation IBC formation. However, in competition experiments between wt and ΔfliC strains, the wt was shown to have a fitness advantage in persisting throughout the urinary tract for 2 weeks, demonstrating a subtle but measurable role for flagella in virulence.

Endolyn (CD164) Interacts with CXCR4 and Modulates the CXCL12-Mediated Homing and Migration of Cord Blood CD133+ Hematopoietic Precusor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1135-1135
Author(s):  
Suzanne M. Watt ◽  
Sinead Forde ◽  
Brit Jorgensen Tye ◽  
Sarah Newey ◽  
Maria Roubelakis
Keyword(s):  
Cord Blood ◽  
Leading Edge ◽  
Class Ii ◽  
Functional Class ◽  
Jurkat Cells ◽  
Temporal Association ◽  
Class Iii ◽  
Human Cd34

Abstract The sialomucin, endolyn or CD164, has been shown to act as an important regulator in the adhesion of human haemopoietic stem/precursor cells (HPC) to stromal niche cells, while also controlling the entry of primitive human CD34+CD38lo HPC into cycle. Here, we define a novel function for endolyn, by identifying its ability to modulate CD133+ cord blood HPC migration on fibronectin towards CXCL12 in vitro. Interestingly, CD133+ cell migration on fibronectin to CXCL12 was reduced 1) by engaging the functional class II glycosylation-dependent epitope on endolyn with the 103B2/9E10 class II but not N6B6 class III antibody; and 2) by RNAi knockdown of endolyn in both CD133+ HPC and Jurkat cells. The inhibition of migration was more pronounced in the more primitive CD34+CD38lo/− HPC subset than in the CD38+ subset. We show a direct and temporal association of endolyn with the CXCR4 receptor, at the leading edge of CD133+ HPC. When CXCL12 is presented on fibronectin, we first see an upregulation in the association of CXCR4 with the a-4 and a-5 integrins that is closely followed by recruitment of endolyn to this complex. This was confirmed by co-immunoprecipitation studies. Knock-down of endolyn using siRNAs revealed that signaling through CXCR4 via PKC-zeta and Akt pathways was significantly dampened, while leaving MAPK phosphorylation unaffected. Our current studies are aimed at examining the in vivo importance of endolyn in HPC homing to the bone marrow of NOD/SCID mice. Our findings support a novel association between three distinct families of cell surface receptors that regulate both cell migratory and proliferative responses and identify endolyn as a key regulator of CXCR4/CXCL12 function.

scholarly journals ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes

1998 ◽  
Vol 142 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Chean Eng Ooi ◽  
Esteban C. Dell'Angelica ◽  
Juan S. Bonifacino
Keyword(s):  
Membrane Trafficking ◽  
Brefeldin A ◽  
Membrane Association ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

scholarly journals MAA-1, a Novel Acyl-CoA–binding Protein Involved in Endosomal Vesicle Transport in Caenorhabditis elegans

2006 ◽  
Vol 17 (10) ◽  
pp. 4318-4329 ◽  
Author(s):  
Morten K. Larsen ◽  
Simon Tuck ◽  
Nils J. Færgeman ◽  
Jens Knudsen
Keyword(s):  
Caenorhabditis Elegans ◽  
Membrane Trafficking ◽  
Binding Protein ◽  
Adaptor Proteins ◽  
Fatty Acyl ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Vesicle Formation

The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydrolysis of acyl-CoA lipid esters. The mechanisms by which these lipid esters are directed to the appropriate membranes in vivo, and their precise roles in vesicle biogenesis, are not yet understood. Here, we present the first report on membrane associated ACBP domain-containing protein-1 (MAA-1), a novel membrane-associated member of the acyl-CoA–binding protein family. We show that in Caenorhabditis elegans, MAA-1 localizes to intracellular membrane organelles in the secretory and endocytic pathway and that mutations in maa-1 reduce the rate of endosomal recycling. A lack of maa-1 activity causes a change in endosomal morphology. Although in wild type, many endosomal organelles have long tubular protrusions, loss of MAA-1 activity results in loss of the tubular domains, suggesting the maa-1 is required for the generation or maintenance of these domains. Furthermore, we demonstrate that MAA-1 binds fatty acyl-CoA in vitro and that this ligand-binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA–dependent process during vesicle formation.

scholarly journals Growth factor gene activation and clonal heterogeneity in an autostimulatory myeloid leukemia.

1989 ◽  
Vol 9 (6) ◽  
pp. 2414-2423 ◽  
Author(s):  
K B Leslie ◽  
J W Schrader
Keyword(s):  
Growth Rate ◽  
Growth Factor ◽  
Leukemia Virus ◽  
Class I ◽  
Class Iii ◽  
Gm Csf ◽  
Rna Transcripts ◽  
Cell Densities

Cell lines were isolated from an in vivo-passaged myelomonocytic leukemia, WEHI-274, that arose in a mouse infected with the Abelson leukemia virus-Moloney leukemia virus complex. Clones were isolated in vitro in the presence or absence of a source of a hemopoietic growth factor, interleukin-3 (IL-3), and were divisible into three distinct classes. All three classes were leukemogenic in vivo. In vitro, the class I clone grew slowly at low cell density but responded with an increased growth rate to IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and autoconditioned medium. Supernatants of these cultures contained a factor with the biological, biochemical, and antigenic properties of IL-3. Class II clones grew better in vitro at low cell densities than did the class I clone and also responded with an increased growth rate to IL-3, GM-CSF, and autoconditional medium but produced GM-CSF rather than IL-3. In contrast, class III clones died in vitro at all cell densities unless exogenous IL-3 or GM-CSF was added. Moreover, they produced no autostimulatory factors. In the class I and class II clones, one allele of the respective IL-3 or GM-CSF gene is rearranged, and in each case, grossly abnormal RNA transcripts of the rearranged gene are present. Neither rearrangements nor abnormal RNA transcripts of the IL-3 or GM-CSF gene were detected in the class III clones. All three classes exhibited a common rearrangement of the c-myb gene, which suggested that all were derived from the one ancestral cell. These experiments demonstrate that two distinct and independent autostimulatory events were involved in the progression of a single disease.

295 ASSESSMENT OF CYTOTOXICITY AND INTERFERENCE OF ATELEIA GLAZIOVIANA IN BOVINE HERPESVIRUS TYPE 1 (BoHV-1) INTERACTION WITH IN VITRO-MATURED BOVINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 304
Author(s):  
D. L. Pavão ◽  
M. M. Piccolomini ◽  
A. C. Góes ◽  
R. Harakava ◽  
M. Haraguchi ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Class Ii ◽  
Class I ◽  
Class Iii ◽  
Herpesvirus Type ◽  
Action Analysis ◽  
Bovine Herpesvirus Type 1 ◽  
Bovine Oocytes ◽  
Class Iv

In vitro embryo production (IVP), as well as having a biotechnical importance, is a valuable tool for studies of gamete and/or embryo interaction with pathogens and xenobiotics. In consequence, it has become an excellent model not only for investigations about sanitary aspects, but also for aspects related to toxic processes. The aim of this study was to evaluate the effect of cytotoxic aqueous extract of Ateleia glazioviana and its interference on the interaction of bovine herpesvirus type 1 (BoHV-1) with bovine oocytes during the In vitro maturation (IVM) period. The statistical analysis of the experiments was made according to Student’s t-test (P < 0.05). The parameters used for this experiment were based on the morphological, physiological, and clastogenic action analysis of the bovine oocytes. The oocytes were collected from ovaries from slaughterhouse and divided into control group (G1, n = 214), a group infected with BoHV-1 (Los Angeles sample 105.5 TCID50 mL-1(G2, n = 210), a group exposed to the extract of A. glazioviana, 0.24 g mL-1; G3, n = 228), and a group simultaneously exposed to the virus and to the extract (G4, n = 210). For IVM, the oocytes were kept in TCM-199 supplemented with hormones and incubated at 38°C, 5%CO2, and 95% humidity for 24 h. The oocytes in G1 showed high expansion of the cumulus cells and ooplasm uniform in appearance; oocytes in G2 showed uniform but moderate expansion of cumulus cells and retraction of ooplasm; the G3 group showed low and irregular expansion with degeneration of cumulus cells and retraction of ooplasm with a granular aspect; and oocytes in G4 showed degeneration of cumulus cells, retracted and granular ooplasm. We observed maturation rates of 81.3% in G1, 31.0% in G2, 5.7% in G3, and 1.4% in G4. As for the clastogenic action analysis, an additional group of oocytes, named in natura (n = 210), was evaluated and presented 41.9% of comets class 0 (zero), 34.8% class I, 12.4% class II, 7.1% class III, and 3.8% class IV G1 (n = 211) presented 6.1% of comets class 0, 47.8% class I, 31.3% class II, 11.0% class III, and 3.8% class IV Oocytes belonging to G3 (217) presented 0.5% of comets class 0, 19.8% class I, 28.1% class II, 34.1% class III, and 17.5% class IV G2 (n = 229) presented 4.4% of comets class 0, 61.2% class I, 26.6% class II, 4.8% class III, and 3.0% class IV Oocytes in G4 (n = 206) presented 3.9% of comets class 0, 26.2% class I, and similar amounts of comets level II (23.8%), III (22.8%), and IV (23.3%). The statistical analysis presented a significant difference in the final results. Such results show the cytotoxic effect of A. glazioviana in bovine oocytes. The simultaneous exposure to the virus and the extract aggravated the effect of the virus, suggesting an increase of the pathogen within the gametic cell. Vitrocel/Embriolife.

scholarly journals Corecognition of HLA-A1 and HLA-DPw3 by a human CD4+ alloreactive T lymphocyte clone.

1990 ◽  
Vol 172 (1) ◽  
pp. 387-390 ◽  
Author(s):  
S Essaket ◽  
J Fabron ◽  
C de Preval ◽  
M Thomsen
Keyword(s):  
T Cell ◽  
Cell Clone ◽  
Class Ii ◽  
Class I ◽  
Mhc Molecules ◽  
T Cell Clone ◽  
Allele Specific ◽  
Antigen Presenting

We have generated an alloreactive proliferative T cell clone that only is stimulated by HLA-DPw3+ antigen presenting cells (APC) that at the same time carry HLA-A1. The T cell clone is CD4+, and the proliferation is blocked by anti-DP monoclonal antibodies and not by antibodies towards other class II or towards class I molecules. Family studies show that APC with A1 and DPw3 on different haplotypes (trans) are able to stimulate the clone, and an HLA recombinant family gives evidence that the class I-carrying part of the haplotype is necessary for stimulation to occur. Stimulation is also observed with mixtures of APC expressing DPw3 and APC expressing A1, and likewise, DPw3+ APC become stimulatory when preincubated with supernatants from A1-positive cells. Our studies suggest that major histocompatibility complex (MHC) class I peptides presented by class II are allostimulatory and that APC can process MHC molecules that presumably are presented as allele-specific peptides in the context of other MHC molecules. We hypothesize that presentation of MHC peptides by MHC molecules constitutes an important part of alloreactive phenomena in vivo and in vitro.

scholarly journals Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay.

1997 ◽  
Vol 17 (8) ◽  
pp. 4611-4621 ◽  
Author(s):  
N Xu ◽  
C Y Chen ◽  
A B Shyu
Keyword(s):  
Mammalian Cells ◽  
Class Ii ◽  
Class I ◽  
Untranslated Regions ◽  
Sequence Motif ◽  
Class Iii ◽  
Decay Kinetics ◽  
Close Proximity

Regulation of cytoplasmic deadenylation has a direct impact on the fate of mRNA and, consequently, its expression in the cytoplasm. AU-rich elements (AREs) found in the 3' untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. AREs direct accelerated deadenylation as the first step in mRNA turnover. Recently we have proposed that AREs can be divided into three different classes. mRNAs bearing either the class I AUUUA-containing ARE or the class III non-AUUUA ARE display synchronous poly(A) shortening, whereas class II ARE-containing mRNAs are deadenylated asynchronously, with the formation of poly(A)- intermediates. In this study, we have systematically characterized the deadenylation kinetics displayed by various AREs and their mutant derivatives. We find that a cluster of five or six copies of AUUUA motifs in close proximity forming various degrees of reiteration is the key feature that dictates the choice between processive versus distributive deadenylation. An AU-rich region 20 to 30 nucleotides long immediately 5' to this cluster of AUUUA motifs can greatly enhance the destabilizing ability of the AUUUA cluster and is, therefore, an integral part of the class I and class II AREs. These two features are the defining characteristics of class II AREs. Our results are consistent with the interpretation that the pentanucleotide AUUUA, rather than the nonamer UUAUUUA(U/A)(U/A), is both an essential and the minimal sequence motif of AREs. Our study provides the groundwork for future characterization of ARE-binding proteins identified by in vitro gel shift assays in order to stringently define their potential role in the ARE-mediated decay pathway. Moreover, transformation of deadenylation kinetics from one type to the other by mutations of AREs implies the existence of cross talk between the ARE and 3' poly(A) tail, which dictates the decay kinetics.

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