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1.
Traffic ; 25(5): e12936, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38725127

RESUMO

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.


Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Endossomos , Fatores de Troca do Nucleotídeo Guanina , Fator de Crescimento Neural , Crescimento Neuronal , Receptor trkA , Animais , Células PC12 , Receptor trkA/metabolismo , Fator de Crescimento Neural/metabolismo , Ratos , Endossomos/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Transporte Proteico , Gânglios Espinais/metabolismo , Camundongos Knockout
2.
PLoS One ; 19(4): e0295103, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38574162

RESUMO

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.


Assuntos
Proteínas Ativadoras de GTPase , Miristatos , Proteínas Ativadoras de GTPase/metabolismo , Mutação Puntual , Ácido Mirístico , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo
3.
Biol Open ; 13(5)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38682696

RESUMO

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Ativadoras de GTPase , Corpos Multivesiculares , Tetraspanina 30 , Tetraspanina 30/metabolismo , Humanos , Corpos Multivesiculares/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Transporte Proteico , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Endossomos/metabolismo , Células HeLa , Proteínas de Transporte
4.
Medicine (Baltimore) ; 103(12): e37549, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38517991

RESUMO

Human umbilical cord mesenchymal stem cells (hUMSCs) belong to a multipotent stem cell population. Transplantation of icariin (ICA)-treated hUMSCs have better tissue repairing function in chronic liver injury. This study was to investigate whether the tissue-repairing effects and migration of hUMSCs after ICA treatment were regulated by circular RNAs (circRNAs). ICA was used to treat hUMSCs in vitro for 1 week and the expression profiles of circRNAs were generated using RNA sequencing. Differentially expressed circRNAs in hUMSCs after ICA intervention were screened. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were carried out to predict the potential function of dysregulated circRNAs. There were 52 differentially expressed circRNAs (32 circRNAs up-regulated and 20 circRNAs down-regulated) with fold change ≥2.0 before and after ICA treatment. ADP-ribosylation factors were associated with the dysregulated circRNAs among Gene Ontology analysis. Kyoto Encyclopedia of Genes and Genomes analysis showed that only endocytosis pathway was associated with up-regulated circRNAs, whereas 4 pathways including homologous recombination, RNA transport, axon guidance, and proteoglycans in cancer were related to down-regulated circRNAs. Fifty-two differentially expressed circRNAs and 238 predicted microRNAs were included in circRNAs-microRNAs network. The mechanism of ICA inducing hUMSCs migration may be through regulating circRNAs expression which affects ADP-ribosylation factors protein signal pathways.


Assuntos
Flavonoides , Células-Tronco Mesenquimais , MicroRNAs , Humanos , RNA Circular/genética , MicroRNAs/genética , Cordão Umbilical , Fatores de Ribosilação do ADP/genética , Perfilação da Expressão Gênica
5.
Arterioscler Thromb Vasc Biol ; 44(5): 1101-1113, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38545783

RESUMO

BACKGROUND: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. METHODS: We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. RESULTS: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. CONCLUSIONS: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.


Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Modelos Animais de Doenças , Resistência à Insulina , Insulina , Camundongos Knockout , Músculo Esquelético , Vasodilatação , Animais , Vasodilatação/efeitos dos fármacos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Insulina/sangue , Masculino , Camundongos Endogâmicos C57BL , Fluxo Sanguíneo Regional , Endotélio Vascular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Densidade Microvascular
6.
FASEB J ; 38(5): e23519, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38457249

RESUMO

ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilium formation. The ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3T31A mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3T31A , ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM-receptor interaction, focal adhesion, and TGF-ß signaling. Similar changes were observed in the proteomic results in ARPE-19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.


Assuntos
Cílios , Proteômica , Humanos , Cílios/genética , Cílios/metabolismo , Transporte Proteico , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Mutação , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo
7.
J Biol Chem ; 300(4): 107124, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38432637

RESUMO

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.


Assuntos
Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento , Proteínas rab de Ligação ao GTP , Humanos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Fator 6 de Ribosilação do ADP , Mutação com Perda de Função , Citocinese/genética , Masculino , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Cílios/genética , Cílios/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Feminino
8.
Nat Commun ; 15(1): 1942, 2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38431634

RESUMO

Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1-Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1-Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1-Gea2 interaction and improves our understanding of molecular events in the membrane trafficking.


Assuntos
Fatores de Ribosilação do ADP , Proteínas de Membrana , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Estrutura Terciária de Proteína , Complexo de Golgi/metabolismo
9.
J Cell Physiol ; 239(4): e31189, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38219074

RESUMO

Joubert syndrome (JBTS) is a systematic developmental disorder mainly characterized by a pathognomonic mid-hindbrain malformation. All known JBTS-associated genes encode proteins involved in the function of antenna-like cellular organelle, primary cilium, which plays essential roles in cellular signal transduction and development. Here, we identified four unreported variants in ARL13B in two patients with the classical features of JBTS. ARL13B is a member of the Ras GTPase family and functions in ciliogenesis and cilia-related signaling. The two missense variants in ARL13B harbored the substitutions of amino acids at evolutionarily conserved positions. Using model cell lines, we found that the accumulations of the missense variants in cilia were impaired and the variants showed attenuated functions in ciliogenesis or the trafficking of INPP5E. Overall, these findings expanded the ARL13B pathogenetic variant spectrum of JBTS.


Assuntos
Anormalidades Múltiplas , Cerebelo/anormalidades , Anormalidades do Olho , Doenças Renais Císticas , Retina/anormalidades , Humanos , Anormalidades Múltiplas/genética , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Anormalidades do Olho/patologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Monoéster Fosfórico Hidrolases/metabolismo , Retina/metabolismo , Cílios/genética , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo
10.
Mol Biol Rep ; 51(1): 106, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38227057

RESUMO

BACKGROUND: ARF (ADP-ribosylation factor) GTPases are major regulators of intracellular trafficking, and classified into 3 groups (Type I - III), among which the type I group members, ARF1 and 3, are responsible genes for neurodevelopmental disorders. METHODS: In this study, we analysed the expression of Type I ARFs ARF1-3 during mouse brain development using biochemical and morphological methods. RESULTS: Western blotting analyses revealed that ARF1-3 are weakly expressed in the mouse brain at embryonic day 13 and gradually increase until postnatal day 30. ARF1-3 appear to be abundantly expressed in various telencephalon regions. Biochemical fractionation studies detected ARF1-3 in the synaptosome fraction of cortical neurons containing both pre- and post-synapses, however ARF1-3 were not observed in post-synaptic compartments. In immunohistochemical analyses, ARF1-3 appeared to be distributed in the cytoplasm and dendrites of cortical and hippocampal neurons as well as in the cerebellar molecular layer including dendrites of Purkinje cells and granule cell axons. Immunofluorescence in primary cultured hippocampal neurons revealed that ARF1-3 are diffusely distributed in the cytoplasm and dendrites with partial colocalization with a pre-synaptic marker, synaptophysin. CONCLUSIONS: Overall, our results support the notion that ARF1-3 could participate in vesicle trafficking both in the dendritic shaft (excluding spines) and axon terminals (pre-synaptic compartments).


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Animais , Camundongos , Fatores de Ribosilação do ADP/genética , Neurônios , Axônios , Cerebelo
11.
J Biomol Struct Dyn ; 42(3): 1268-1279, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37078745

RESUMO

IQSEC2 gene mutations are associated with epilepsy, autism, and intellectual disability. The primary function IQSEC2, mediated via its Sec 7 domain, is to act as a guanine nucleotide exchange factor for ARF6. We sought to develop a molecular model, which may explain the aberrant Sec 7 activity on ARF6 of different human IQSEC2 mutations. We integrated experimental data of IQSEC2 mutants with protein structure prediction by the RaptorX server combined with molecular modeling and molecular dynamics simulations. Normally, apocalmodulin (apoCM) binds to IQSEC2 resulting in its N-terminal fragment inhibiting access of its Sec 7 domain to ARF6. An increase in Ca2+ concentration destabilizes the interaction of IQSEC2 with apoCM and removes steric hindrance of Sec 7 binding with ARF6. Mutations at amino acid residue 350 of IQSEC2 result in loss of steric hindrance of Sec 7 binding with ARF6 leading to constitutive activation of ARF6 by Sec 7. On the other hand, a mutation at amino acid residue 359 of IQSEC2 results in constitutive hindrance of Sec 7 binding to ARF6 leading to the loss of the ability of IQSEC2 to activate ARF6. These studies provide a model for dysregulation of IQSEC2 Sec 7 activity by mutant IQSEC2 proteins.Communicated by Ramaswamy H. Sarma.


Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutação , Modelos Moleculares , Aminoácidos/genética
12.
Int J Biol Macromol ; 254(Pt 2): 127898, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37939768

RESUMO

The ADP ribosylation factor like protein 15 (ARL15) gene encodes for an uncharacterized GTPase associated with rheumatoid arthritis (RA) and other metabolic disorders. Investigation of the structural and functional attributes of ARL15 is important to position the protein as a potential drug target. Using spectroscopy, we demonstrated that ARL15 exhibits properties inherent of GTPases. The Km and Vmax of the enzyme were calculated to be 100 µM and 1.47 µmole/min/µL, respectively. The equilibrium dissociation constant (Kd) of GTP binding with ARL15 was estimated to be about eight-fold higher than that of GDP. Small Angle X-ray Scattering (SAXS) data indicated that in solution, the apo state of monomeric ARL15 adopts a shape characterized by a globe of maximum linear dimension (Dmax) of 6.1 nm, and upon binding to GTP or GDP, the vector distribution profile changes to peak-n-tail shoulder with Dmax extended to 7.6 and 7.7 nm, respectively. Structure restoration using a sequence-based template and experimental SAXS data provided the first visual insight revealing that the folded N-terminal in the unbound state of the protein may toggle open upon binding to guanine nucleotides. The conformational dynamics observed in the N-terminal region offer a scope to develop drugs that target this unique GTPase, potentially providing treatments for a range of metabolic disorders.


Assuntos
Artrite Reumatoide , Doenças Metabólicas , Humanos , Nucleotídeos de Guanina , Nucleotídeos/metabolismo , Guanina , Espalhamento a Baixo Ângulo , Difração de Raios X , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Difosfato
13.
Plant Physiol ; 194(2): 673-683, 2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-37787604

RESUMO

Polarity of plasma membrane proteins is essential for cell morphogenesis and control of cell division and, thus, influences organ and whole plant development. In Arabidopsis (Arabidopsis thaliana) root endodermal cells, 2 transmembrane kinases, INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) and KINASE ON THE INSIDE (KOIN), accumulate at opposite lateral domains. Their polarization is tightly linked to their activities regulating cell division and ground tissue patterning. The polarization of IRK and KOIN relies solely on the secretion of newly synthesized protein. However, the secretion machinery by which their opposite, lateral polarity is achieved remains largely unknown. Here, we show that different sets of ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factors (ARF-GEFs) mediate their secretion. ARF-GEF GNOM-like-1 (GNL1) regulates KOIN secretion to the inner polar domain, thereby directing KOIN sorting early in the secretion pathway. For IRK, combined chemical and genetic analyses showed that the ARG-GEF GNL1, GNOM, and the BREFELDIN A-INHIBITED-GUANINE NUCLEOTIDE-EXCHANGE FACTORs 1 to 4 (BIG1-BIG4) collectively regulate its polar secretion. The ARF-GEF-dependent mechanisms guiding IRK or KOIN lateral polarity were active across different root cell types and functioned regardless of the protein's inner/outer polarity in those cells. Therefore, we propose that specific polar trafficking of IRK and KOIN occurs via distinct mechanisms that are not constrained by cell identity or polar axis and likely rely on individual protein recognition.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Arabidopsis/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo
14.
Curr Protein Pept Sci ; 25(2): 137-153, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37718516

RESUMO

ARL15 is a member of the RAS superfamily of small GTPases and is associated with several metabolic traits, including increased risk of diabetes, rheumatoid arthritis and lipid metabolism disorders. The ARL15 gene encodes for an uncharacterized small GTP binding protein. Its precise role in human physiology remains unknown, but several genetic association studies have recognized different variants in this gene to be statistically associated with numerous traits and complex diseases. Here, we provided the unique features of ARL15 small G protein, its association with varied metabolic and lifestyle diseases, its function in vesicular and lipid trafficking, and its binding partners. We outlined this protein as a promising and emerging therapeutic target to combat metabolic disorders like cardiovascular diseases, diabetes and rheumatoid arthritis. The review provides a comprehensive description of the current advancements in ARL15 research with a perspective that focused research will position this small GTPase as a viable target for the treatment of rheumatoid arthritis.


Assuntos
Artrite Reumatoide , Diabetes Mellitus , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Estudos de Associação Genética , Fenótipo
15.
Brain ; 147(5): 1751-1767, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38128568

RESUMO

BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T>C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A>C (p.Thr66Pro) and c.124T>C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system.


Assuntos
Lisossomos , Doenças Neurodegenerativas , Humanos , Lisossomos/metabolismo , Lisossomos/genética , Feminino , Masculino , Doenças Neurodegenerativas/genética , Animais , Lactente , Pré-Escolar , Criança , Peixe-Zebra , Linhagem , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Alelos , Mutação de Sentido Incorreto/genética
16.
eNeuro ; 10(11)2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37848288

RESUMO

During the development of the cerebral cortex, N-cadherin plays a crucial role in facilitating radial migration by enabling cell-to-cell adhesion between migrating neurons and radial glial fibers or Cajar-Reztius cells. ADP ribosylation factor 4 (Arf4) and Arf5, which belong to the Class II Arf small GTPase subfamily, control membrane trafficking in the endocytic and secretory pathways. However, their specific contribution to cerebral cortex development remains unclear. In this study, we sought to investigate the functional involvement of Class II Arfs in radial migration during the layer formation of the cerebral cortex using mouse embryos and pups. Our findings indicate that knock-down of Arf4, but not Arf5, resulted in the stalling of transfected neurons with disorientation of the Golgi in the upper intermediate zone (IZ) and reduction in the migration speed in both the IZ and cortical plate (CP). Migrating neurons with Arf4 knock-down exhibited cytoplasmic accumulation of N-cadherin, along with disturbed organelle morphology and distribution. Furthermore, supplementation of exogenous N-cadherin partially rescued the migration defect caused by Arf4 knock-down. In conclusion, our results suggest that Arf4 plays a crucial role in regulating radial migration via N-cadherin trafficking during cerebral cortical development.


Assuntos
Caderinas , Neurônios , Animais , Camundongos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Movimento Celular/fisiologia , Córtex Cerebral/metabolismo , Complexo de Golgi/metabolismo , Neurônios/metabolismo
17.
Int J Mol Sci ; 24(19)2023 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-37834383

RESUMO

Despite the "big data" on cancer from recent breakthroughs in high-throughput technology and the development of new therapeutic modalities, it remains unclear as to how intra-tumor heterogeneity and phenotypic plasticity created by various somatic abnormalities and epigenetic and metabolic adaptations orchestrate therapy resistance, immune evasiveness, and metastatic ability. Tumors are formed by various cells, including immune cells, cancer-associated fibroblasts, and endothelial cells, and their tumor microenvironment (TME) plays a crucial role in malignant tumor progression and responses to therapy. ADP-ribosylation factor 6 (ARF6) and AMAP1 are often overexpressed in cancers, which statistically correlates with poor outcomes. The ARF6-AMAP1 pathway promotes the intracellular dynamics and cell-surface expression of various proteins. This pathway is also a major target for KRAS/TP53 mutations to cooperatively promote malignancy in pancreatic ductal adenocarcinoma (PDAC), and is closely associated with immune evasion. Additionally, this pathway is important in angiogenesis, acidosis, and fibrosis associated with tumor malignancy in the TME, and its inhibition in PDAC cells results in therapeutic synergy with an anti-PD-1 antibody in vivo. Thus, the ARF6-based pathway affects the TME and the intrinsic function of tumors, leading to malignancy. Here, we discuss the potential mechanisms of this ARF6-based pathway in tumorigenesis, and novel therapeutic strategies.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Células Endoteliais/metabolismo , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/genética , Microambiente Tumoral , Neoplasias Pancreáticas
18.
Microcirculation ; 30(8): e12831, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37750425

RESUMO

OBJECTIVE: Endocytosis is a process vital to angiogenesis and vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of blunting endocytic processes have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for clathrin-mediated and clathrin-independent endocytosis. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide an analysis of Arf6's function in angiogenic endothelium, focusing on its role in actin and endocytosis as well as sprouting morphogenesis. METHODS: Primary endothelial cells were cultured in both 2D and 3D environments. Here, endothelial cells were fixed and stained for various proteins or transfected with fluorescently-tagged constructs for live-cell imaging. RESULTS: We found that Arf6 localized to both filamentous actin and sites of endocytosis in two-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, which may be the primary driver underlying gross sprouting dysmorphogenesis in its absence. CONCLUSIONS: Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and endocytosis and is required for proper sprout formation.


Assuntos
Fator 6 de Ribosilação do ADP , Actinas , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Células Endoteliais/metabolismo , Endocitose/fisiologia , Clatrina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular
19.
DNA Cell Biol ; 42(10): 617-637, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37610843

RESUMO

Recent studies have shown that several members of the G-protein-coupled receptors (GPCR) superfamily play crucial roles in the maintenance of ion-water homeostasis of the sperm and Sertoli cells, development of the germ cells, formation of the blood barrier, and maturation of sperm. The GPCR, guanyl-nucleotide exchange factor, membrane traffic protein, and small GTPase genes were analyzed by microarray and bioinformatics (3513 sperm and Sertoli cell genes). In the microarray analyses of three human cases with different nonobstructive azoospermia sperm, the expression of GOLGA8IP, OR2AT4, PHKA1, A2M, OR56A1, SEMA3G, LRRC17, APP, ARHGAP33, RABGEF1, NPY2R, GHRHR, LTB4R2, GRIK5, OR6K6, NAPG, OR6C65, VPS35, FPR3, and ARL4A was upregulated, while expression of MARS, SIRPG, OGFR, GPR150, LRRK1, and NGEF was downregulated. There was an increase in GBP3, GBP3, TNF, TGFB3, and CLTC expression in the Sertoli cells of three human cases with NOA, whereas expression of PAQR4, RRAGD, RAC2, SERPINB8, IRPB1, MRGPRF, RASA2, SIRPG, RGS2, RAP2A, RAB2B, ARL17, SERINC4, XIAP, DENND4C, ANKRA2, CSTA, STX18, and SNAP23 were downregulated. A combined analysis of Enrich Shiny Gene Ontology (GO), STRING, and Cytoscape was used to predict proteins' molecular interactions and then to recognize master pathways. Functional enrichment analysis showed that the biological process (BP), regulation of protein metabolic process, regulation of small GTPase-mediated signal transduction were significantly expressed in up-/downregulated differentially expressed genes (DEGs) in sperm. In molecular function (MF) experiments of DEGs that were up-/downregulated, it was found that GPCR activity, guanyl ribonucleotide binding, GTPase activity and nucleoside-triphosphatase activity were overexpressed. An analysis of GO enrichment findings of Sertoli cells showed BP and MF to be common DEGs. When these gene mutations have been validated, they can be used to create new GPCR antagonists or agonists that are receptor-selective.


Assuntos
Azoospermia , Proteínas Monoméricas de Ligação ao GTP , Humanos , Masculino , Testículo/metabolismo , Azoospermia/genética , Azoospermia/metabolismo , Sêmen/metabolismo , Expressão Gênica , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/genética , Anquirinas/genética , Anquirinas/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismo
20.
Proc Natl Acad Sci U S A ; 120(34): e2302603120, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37579161

RESUMO

Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway, indispensable for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. Murine Rab-like 2 (Rabl2) GTPase resembles Chlamydomonas Arf-like 3 (ARL3) GTPase in promoting outward TZ passage of the signaling protein cargo-laden BBSome. During this process, ARL3 binds to and recruits the retrograde IFT train-dissociated BBSome as its effector to diffuse through the TZ for ciliary retrieval, while how RABL2 and ARL3 cross talk in this event remains uncertain. Here, we report that Chlamydomonas RABL2 in a GTP-bound form (RABL2GTP) cycles through cilia via IFT as an IFT-B1 cargo, dissociates from retrograde IFT trains at a ciliary region right above the TZ, and converts to RABL2GDP for activating ARL3GDP as an ARL3 guanine nucleotide exchange factor. This confers ARL3GTP to detach from the ciliary membrane and become available for binding and recruiting the phospholipase D (PLD)-laden BBSome, autonomous of retrograde IFT association, to diffuse through the TZ for ciliary retrieval. Afterward, RABL2GDP exits cilia by being bound to the ARL3GTP/BBSome entity as a BBSome cargo. Our data identify ciliary signaling proteins exported from cilia via the RABL2-ARL3 cascade-mediated outward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defect-induced Bardet-Biedl syndrome caused by RABL2 mutations in humans could be well explained in a mutation-specific manner, providing us with a mechanistic understanding behind the outward BBSome TZ passage required for proper ciliary signaling.


Assuntos
Cílios , Proteínas Hedgehog , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/genética , Proteínas rab de Ligação ao GTP/metabolismo , Chlamydomonas
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