Rozprawy doktorskie na temat „Arl14”

Two closely related human Arls, Arl8a and Arl8b, were found to localise to lysosomes in mammalian cells. conventionally, membrane binding of Arf and Arl proteins is mediated by both an N-terminal myristoyl group and an N-terminal amphipathic helix that are inserted into the lipid bilayer upon activation of the GTPase. Arl8 GPTases lack myristolylation sites, and examination of the N-terminus of Arl8b revealed that it contains an acetyl group instead, and this acetylated methionine is necessary for its lysosomal location. Lysosomes of cells overexpressing Arl8b move more frequently, suggesting a role for Arl8a and Arl8b as positive regulators of lysosomal transport. Arl4a, Arl4c and Arl4d are very similar in sequence and were found to act in a pathway upstream of Arf6, Arf6 is a regulator of key processes at the plasma membrane, such as endocytosis, actin dynamics and cell adhesion. One of the major activators of Arf6 is the exchange factor ARNO (‘Arf nucleotide binding site opener’). In order to activate Arf6, ARNO must translocate from the cytoplasm onto the plasma membrane. ARNO was identified as an interaction partner of Arl4 GTPases in a yeast two hybrid screen, and Arl4 GTPases mediate membrane translocation of ARNO. Thus, Arl4 GTPases represent a novel signal transduction pathway upstream of Arf6. This study provides a characterisation of members of the human Arl family and suggests functions for Arl4 and Arl8 GTPases in membrane traffic related processes.

Zebrafish has emerged as an important vertebrate model for studying hematopoiesis and its genetic and chemical modifiers. The zebrafish embryos are unique in their optical transparency, ease of maintenance and high fecundity. They are also amendable to genetic and pharmacological perturbation at high throughput. As a result, the embryos are suitable for various experimental techniques and have a high efficiency in large-scale drug screening. Recently, zebrafish has also emerged as a model for the study of human disease. In this model organism, primitive hematopoiesis is transitory and it occurs in the intermediate cells mass and comprises primarily erythroid cells. Definitive hematopoiesis arises from the ventral wall of dorsal aorta and moves to the caudal hematopoietic tissues, thence the kidney, where life-long and multi-lineage differentiation occurs. The chemical screening platform comprises O-dianisidine staining for hemoglobin containing cells (erythroid) during primitive hematopoiesis. Positive hits were validated based on flow cytometry of dissociated transgenic Tg(gata1:GFP) embryos and whole-mount in-situ hybridization (WISH) for hematopoietic genes. Gene knock-down was conducted by morpholinos (MO) injected into zebrafish embryos at 1-4 cell stage and the effects on hematopoietic development evaluated by WISH and quantitative real-time PCR. Chemical screening of 74 compounds has been performed. These compounds were obtained from a chemical library comprising 879 compounds from NIH (National Institutes of Health) and pre-screened by their effects on cancer cell lines. Four compounds (Tin(IV), chlorotriphenyl [1-(4-ethoxyphenyl)- 3-cyanoureato]-hydrogen,triethylamine, Nogamycin, N,N-Dibenzyldaunorubicin hydrochloride and Allyl 2,3,4-tri-O-benzyl-6-O-(tert-butyldimethylsilyl)-α- D-gluco-Pyranoside) which significantly reduced O-dianisidine staining were identified of which one (Allyl 2,3,4-tri-O-benzyl-6-O-(tert-butyldimethylsilyl) -α-D-glucopyranoside was shown to reduce GFP+ population in Tg(gata1:GFP) population Another chemical (2-Propanol,1,1'-[(1-methylethylidene)bis(4, 1-phenyleneoxy)]bis[3-[(1,1,3,3-tetramethylbutyl)amino]-,dihydrochloride]) was shown to reduce c-myb (marker of definitive hematopoiesis) expression in the ventral wall of dorsal aorta. I also attempted gene knock in zebrafish embryos based on anti-sense morpholino microinjection. A gene encoding for arl4ab was examined, as it was shown to be expressed in hematopoietic tissue in zebrafish embryos but its function is entirely unknown. Knock-down of arl4ab significantly reduced c-myb and runx1 expression in the ventral wall of dorsal aorta and it can be reversed by co-injecting arl4ab mRNA. scl and gata1 expression as well as GFP expression in transgenic Tg(flk1:GFP) embryos that represented vascular development was unaffected. In summary, a zebrafish platform for the study of chemical and genetic modifiers was established. The results have provided important leads for further study into the mechanisms whereby these modifiers regulate hematopoiesis in the zebrafish embryos.
published_or_final_version
Medicine
Master
Master of Medical Sciences

碩士
臺灣大學
分子醫學研究所
98
ARF-like (ARL) proteins belong to ADP-ribosylation factor (ARF) GTPase family, which are involved in protein trafficking and cytoskeleton organization. Those small G proteins require guanine-nucleotide exchange factors (GEFs) to switch from GDP-bound to GTP-bound form and become active. Previous reports suggested that Syt1p is the GEF of Arf2p and is involved in vesicle trafficking. Recently, our studies showed that Syt1p also acted as a GEF for Arl1p. In this study, Syt1p was further characterized. Firstly, it has been shown that Syt1p belongs to BFA-resistant GEFs. Secondly, Syt1p can use multiple regions to interact with Arl1p. The N-terminus, Sec7 domain, and C-terminus of Syt1p can all interact with Arl1d17N form, whose N-terminal first 17 amino acids are deleted. The interactions between all of the three regions and Arl1d17N are stronger than the interaction between full-length Syt1p and Arl1pd17N. Therefore, it might hint that Syt1p is autoregulated as other GEFs of Small GTPases. Surprisingly, we next found that Syt1p has an intramolecular interaction between the C-terminal region and Sec7 domain and an intermolecular interaction between C-terminal regions, indicating that Syt1p could form dimers or oligomers and might be autoregulated. Syt1p dimerization or oligomerization is also supported by in vivo pull down results, which proved that Syt1p can interact with itself. Moreover, the N-terminus is important for the formation of dimers or oligomers. Yeast two-hybrid screen was also performed to search for putative regulators of Syt1p. However, those candidates remain to be elucidated. Besides, whether dimerization or oligomerization plays an important role in Syt1p activation or membrane tethering and whether autoinhibition truly exists in Syt1p in vivo require further investigation.

Influenza A virus (IAV) is an important human pathogen that causes epidemic and pandemic events of flu. The study of the viral life cycle and its interactions with the infected host is crucial for the development of novel therapeutic strategies. IAV has an eight-part segmented RNA genome organized in viral ribonucleoprotein complexes (vRNP), which are replicated in the nucleus of the cell. De novo synthesized vRNPs need to leave the nucleus to reach the cytosol for viral assembly, budding and release. Several pathways have been implicated in nuclear export of vRNPs, including CRM1, apoptosis activation and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling cascade. Mitochondria are crucial organelles for the maintenance of cellular homeostasis, since they are responsible for the regulation of metabolism, apoptosis, calcium homeostasis and innate immunity. Their functions are tightly regulated by dynamic changes in mitochondrial morphology. Given their importance, many viruses modulate mitochondria to promote cellular environments favoring their proliferation. IAV has been shown to fragment mitochondria to decrease the antiviral immune response. Our lab identified a candidate modulator of mitochondrial morphology and IAV infection: the host GTPase Arl17. Our work demonstrated that depletion of Arl17 leads to a reduction in viral titers and promotes mitochondrial fragmentation, regardless of infection. Interestingly, this phenotype was not accompanied by alterations in IFNβ1 expression and mitochondrial unfolded protein response activation (UPRmt). However, ATP levels were significantly reduced in the absence of Arl17. Additionally, we showed that Arl17 is required for regular vRNP nuclear export. In its absence, we observed a delay in vRNPs nuclear export that was CRM1- and ERK1/2-independent. Therefore, influence of Arl17 on the induction of apoptosis should be further investigated as its inhibition could explain vRNPs nuclear export delay and it could be the element that links mitochondria and vRNPs nuclear export.

碩士
國立臺灣大學
分子醫學研究所
106
Arf-like proteins (Arls) are important regulators involved in a diversity of biological events, especially vesicle trafficking. In Saccharomyces cerevisiae, Arl1 acts to recruit a golgin protein Imh1 to the trans-Golgi network (TGN). However, besides the role of Imh1 as a high-copy suppressor of Ypt6, an important Rab protein mediating endosome-to-Golgi trafficking, less is known about it physiological significance. In previous studies (Hsu et al., 2016), they demonstrated that the Unfolded-Protein Response (UPR) augmented the activity of Arl1 and the subsequent Imh1 recruitment, but it remained to be elucidated on its specific functions under ER stress. Here, we showed that the UPR-activated Arl1 and Imh1 act to specifically maintain the endosome-to-Golgi trafficking when cell encounters ER stress. Moreover, this regulation does not simply depend on GARP complex recruitment, an important tethering complex, to affect retrograde trafficking. We found that the first five amino acids at Imh1 N-terminus is required for its function, as it contributes to the recruitment of Sft2, a tetra-spanning membrane protein involved in vesicle fusion. The N-terminus of Sft2 is responsible for facilitating the transport of Snc1 and Tlg1 and promoting retrograde trafficking. Together, our study is one of the first to demonstrate the physiological function of golgin Imh1 and elucidate the importance of the trafficking machinery in response to ER stress.

碩士
國立臺灣大學
分子醫學研究所
94
ADP-ribosylation factors (ARFs) and ARF-like proteins (ARLs) are members of the ARF family, which play essential roles in intracellular membrane trafficking. These proteins are regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) to cycle between active GTP-bound form and inactive GDP-bound form. ARLs are ~40–60 % identical to each other and to ARFs. Despite their high similarity to ARFs, only ARL1 has been shown to be involved in vesicular transport. ARL1 is localized in the Golgi complex and may participate in trans-Golgi network (TGN) to plasma membrane or endosome to TGN trafficking pathway. In the first part, we investigated the function of ARL1 by using constitutively active (Q71L) or inactive (T31N) ARL1. Discrepant with previous studies, we found that overexpressed ARL1Q71L specifically disperses Golgin-245 from Golgi apparatus. Over-expressed ARL1T31N disrupted trans-Golgi proteins but not cis/medial Golgi and endosome proteins. Moreover, VSVG transport was blocked in ARL1Q71L overexpressed but not in ARL1 knockdown cells which implied that ARL1 itself may not directly participate in the VSVG transport. In the second part, we attempted to find possible GAP for ARL1. Among our three candidates, ARFGAP1 did not interact with ARL1 in yeast two-hybrid assay but endogenous ARL1 were dissociated from the Golgi in cells with overexpressed ARFGAP1, which we considered an indirect consequence of general effect on Golgi proteins. Centaurin α1 neither interacted with ARL1 nor affected ARL1 localization. ARAP1 interacted with ARL1Q71LdN, but we could not detect the change of ARL1 localization when ARAP1 overexpressed. Whether ARAP1 could be GAP for ARL1 needs to be investigated further. In the third part, we identified an ARL1Q71L interacting protein, CIB1. We showed that ARL1 interacted with CIB1 in a Ca2+-dependent manner. Furthermore, CIB1 was ubiquitously expressed in cells especially colocalized with ARL1 in the Golgi apparatus in particular conditions. In addition, Golgi localization of CIB1 was not dependent on ARL1, and vice versa. Unfortunately, even though the interaction and colocalization of ARL1 and CIB1 were observed, we could not demonstrate the functional relevance between these two proteins.

博士
長庚大學
生物醫學研究所
103
The small GTPase Arf-like protein 1 (Arl1) is well known for its role in intracellular vesicular transport at the trans-Golgi network (TGN). In here, we used differential affinity chromatography combined with mass spectrometry to identify Arf-interacting protein 1 (including arfaptin-1a and arfaptin-1b) as an Arl1-interacting protein. Yeast two-hybrid demonstrated that arfaptin-1 directly interacted with Arl1 through their arfaptin homolog (AH) domain. Arl1 knockdown caused arfaptin-1 dispersed from the Golgi apparatus. Expression of the wild-type Arl1, but not Arl1TN or wild-type Arf1 in Arl1- knockdown cells restored the Golgi localization of arfaptin-1. The Golgi dissociations of arfaptin-1 and Arl1 were temporally coupled in response to brefeldin-A treatment. These results demonstrated that Arl1 medicates the Golgi targeting of arfaptin-1. We also characterized a novel function for arfaptin-1 in Arl1-mediated retrograde transport. Using a Shiga-toxin subunit B (STxB) transportation assay, we demonstrated that knockdown of arfaptin-1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin-1 overexpression, but not an Arl1 binding-defective mutant (arfaptin-1b-F317A), consistently inhibited STxB transport. Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa. Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport. Our results show that arfaptin-1 is an Arl1 effector and acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.

碩士
國立臺灣大學
藥理學研究所
94
Abstract The discovery of a fourth member of the opioid receptor family, opioid receptor-like (ORL-1) recptor in 1994 gave rise to a new camp in opioid research, since this new G-protein coupled receptor though being in logically homogenous to classical opioid receptors’ did not bind opiates with high affinity. A year later, two groups independently identified the endogenous ligand of this receptor, a 17 amino acid neuropeptide named nociceptin or orphanin FQ. This receptor family was officially renamed after its endogenous ligand as nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) receptor in IUPHAR2002. The NOP receptor and N/OFQ are widely distributed in the central nervous system (CNS), and the periphery. Several studies have implicated the N/OFQ-NOP receptor plays an important role system in pain, anxiety, learning, memory, food intake, diuresis, and drug addition. However, N/OFQ modulation of pain pathways is yet to be completely understood. N/OFQ, unlike traditional opioids which display analgesia effect, elicited both analgesia and hyperalgesia. It has great benefit in understanding the physiological role(s) of N/OFQ in pain regulation by developing NOP receptor ligands, including agonists and antagonists. A novel ligand for the receptor (NOP), [(pF)Phe4, Arg14, Lys15] N/OFQ-NH2 (UFP-102), has been generated by combining in the N/OFQ-NH2 sequence two chemical modifications, [Arg14, Lys15] and [(pF)Phe4], that have been previously demonstrated to increase potency and ability lasting, respectively. The present study, quantitatively investigated the potency of UFP-102 at NOP receptors of rat brain slices containing the midbrain ventrolateral periaquaductal gray (vlPAG), a crucial site for N/OFQ-induced reversal of opioid algesia, using the blind patch-clamp whole cell recording technique. UFP-102 concentration-dependently induced an outward current at resting membrane potential and increased the membrane current elicited by hyperpolarization ramps from -60 to -140 mV in ventrolateral PAG neurons. The current induced by UFP-102 is characterized with inward rectification and has reversal potential near the equilibrium potential of K+ ions according to the Nernst equation. Therefore, UFP-102 activates inwardly rectifying K+ channels. UFP-102 showed similar maximal effects (35 ± 3% and 40 ± 4%, respectively) but higher potency (4- to 5-fold) relative to N/OFQ. UFP-101, a novel selective and competitive antagonist of NOP receptors, concentration-dependently attenuated of K+ current induced by UFP-102 but did not change its reversal potential. It produced a parallel right-shift of the concentration-response curve of UFP-102 but did not alter the extent of UFP-102 induced maximal response. The antagonistic effect of UFP-101 on UFP-102 induced K+ current was surmountable by increasing the UFP-102 concentrations. However, the nonselective opioid receptor antagonist, naloxone, did not attenuated of K+ current induced by UFP-102. The pIC50 of UFP-101 against UFP-102 was 6.52. It is concluded that, in ventrolateral PAG, (1) UFP-102 is a selective agonist of NOP receptor with an EC50 value of 11±2 nM. (2) UFP-102 showed similar maximal effects but higher potency (about 4- to 5-fold) relative to N/OFQ.

Les protéines DOCK180 et ELMO coopèrent ensemble biochimiquement et génétiquement afin d’activer la GTPase Rac1 lors de plusieurs évènements biologiques. Toutefois, le rôle que jouent ces protéines dans la signalisation par Rac est encore mal compris. Nous émettons l’hypothèse que Dock180 agit comme activateur de Rac, alors que ELMO est requis pour l’intégration de la signalisation de Rac plutôt que son activation per se. Nous postulons que ELMO agit comme signal de localisation intracellulaire afin de restreindre de façon spatio-temporelle la signalisation de Rac en aval de Dock180, et/ou que ELMO agit comme protéine d’échafaudage entre Rac et ses effecteurs pour amplifier la migration cellulaire. Dans l’objectif nº 1, nous démontrons que le domaine PH atypique de ELMO1 est le site d’interaction principal entre cette protéine et DOCK180. De plus, nous démontrons que la liaison entre ELMO et DOCK180 n’est pas nécessaire pour l’activation de Rac, mais est plutôt essentielle pour faciliter la réorganisation du cytosquelette induite par l’activation de Rac en aval de Dock180. Ces résultats impliquent que ELMO pourrait jouer des rôles additionnels dans la signalisation par Rac. Dans l’objectif nº 2, nous avons découvert l’existence d’une homologie structurelle entre ELMO et un module d’autorégulation de la formine Dia1, et avons identifié trois nouveaux domaines dans la protéine ELMO : les domaines RBD, EID et EAD. De façon analogue à Dia1, nous avons découvert que ELMO à l’état basal est autoinhibé grâce à des intéractions intramoléculaires. Nous proposons que l’état d’activation des protéines ELMO est régulé de façon similaire aux formines de la famille Dia, c’est-à-dire grâce à des interactions avec d’autres protéines. Dans l’objectif nº 3, nous identifions un domaine RBD polyvalent chez ELMO. Ce domaine possède une double spécificité pour les GTPases de la famille Rho et Arf. Nous avons découvert que Arl4A agit comme signal de recrutement membranaire pour le module ELMO/DOCK180/Rac. Nos résultats nous permettent de supposer que d’autres GTPases pourraient être impliquées dans l’activation et la localisation de cette voie de signalisation. Nous concluons qu’à l’état basal, ELMO et DOCK180 forment un complexe dans lequel ELMO est dans sa conformation autoinhibée. Bien que le mécanisme d’activation de ELMO ne soit pas encore bien compris, nous avons découvert que, lorsqu’il y a stimulation cellulaire, certaines GTPases liées au GTP peuvent intéragir avec le domaine RBD de ELMO pour relâcher les contacts intramoléculaires et/ou localiser le complexe à la membrane. Ainsi, les GTPases peuvent servir d’ancrage au complexe ELMO/DOCK180 pour assurer une regulation spatiotemporelle adequate de l’activation et de la signalisation de Rac.
DOCK180 and ELMO cooperate biochemically and genetically to activate Rac in several biological events. However, the role of these proteins in Rac signaling is still poorly understood. We hypothesize that DOCK180 functions as a RacGEF, with ELMO binding to DOCK180 being required for integration of proper Rac signaling rather than Rac activation per se. We postulate that ELMO acts as a subcellular targeting signal for spatio-temporal restriction of DOCK180-mediated Rac signaling and/or as a scaffold for Rac effectors to enforce cell migration. In Aim #1, we elucidate that the atypical ELMO1 PH is the major DOCK180 binding site. We demonstrate that the binding of ELMO1 to DOCK180 is not necessary for Rac GTP-loading, but is instead required to facilitate Rac-GTP induced cytoskeletal changes following DOCK180 activation. These results imply additional roles for ELMO in mediating Rac signaling. In Aim #2, we reveal structural homology between ELMO and an autoregulatory module in the formin, Dia1, and identify three novel domains in ELMOs: the RBD, EID and EAD. Analogous to Dia1, we uncovered that ELMO is autoinhibited via intramolecular interactions at basal state. We propose that the activation state of ELMO proteins is regulated, much like in Dia-family formins, via interaction with other proteins. Aim #3 identifies a polyvalent RBD in ELMO with dual specificity for Rho and Arf family GTPases. We found Arl4A as a novel membrane recruitment signal for the ELMO/DOCK180/Rac module. Our results may have broad implications in the activation and localization of this pathway by additional GTPases. We conclude that, at basal levels, ELMO/DOCK180 is complexed, with ELMO in an autoinhibited state in the cytosol. Through cell stimulation, certain GTPases will be activated that now bind the ELMO RBD and alleviate the intramolecular contacts. In this way, the GTPase anchors the activated ELMO/DOCK180 module in place for proper spatio-temporal regulation of Rac activation and signaling.