RESUMO
Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Proteínas de Membrana Transportadoras , Apoproteínas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ftalimidas/metabolismo , Conformação Proteica , Especificidade por SubstratoRESUMO
The dopamine transporter (DAT) is closely related to a variety of neurological disorders including Parkinson's disease (PD) and other neurodegenerative diseases. In vivo imaging of DAT with radio-labelled tracers has become a powerful technique in related disorders. The radioiodine-labelled tropane derivative [123I]FP-CIT ([123I]1a) is widely used in clinical single photon emission computed tomography (SPECT) imaging as a DAT imaging agent. To develop more metabolically stable DAT radioligands for accurate imaging, this work compared two novel deuterated tropane derivatives ([131I]1c-d) with non-deuterated tropane derivatives ([131I]1a-b). [131I]1a-d were obtained in high radiochemical purity (RCP) above 99 % with molar activities of 7.0-10.0 GBq/µmol. The [131I]1a and [131I]1c exhibited relatively higher affinity to DAT (Ki: 2.0-3.12 nM) than [131I]1b and [131I]1d. Biodistribution results showed that [131I]1c consistently exhibited a higher ratio of the target to non-target (striatum/cerebellum) than [131I]1a. Furthermore, metabolism studies indicated that the in vivo metabolic stability of [131I]1c was superior to that of [131I]1a. Ex vivo autoradiography showed that [131I]1c selectively localized on DAT-rich striatal regions and the specific signal could be blocked by DAT inhibitor. These results indicated that [131I]1c might be a potential probe for DAT SPECT imaging in the brain.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina , Radioisótopos do Iodo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Radioisótopos do Iodo/metabolismo , Distribuição Tecidual , Tropanos , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
In this manuscript, the generation of an optical vortex beam with high order and reconfigurable orbital angular momentum (OAM) is studied. Multi-waveguide holographic gratings (MWHG) are deployed to generate OAM beams with high order. The generation of the OAM beam with an order l from +4 to +8 is demonstrated by numerical simulations, and the generated OAM order is manipulable and configurable by incident phase. The working bandwidths of the MWHG for different OAM orders are at the level of 40 nm. This work could provide valuable references for practical implementation of OAM in integrated optics.
RESUMO
The secretion and quality control of glycosylphosphatidylinositol-anchored proteins (GPI-APs) necessitates post-attachment remodeling initiated by the evolutionarily conserved PGAP1, which deacylates the inositol in nascent GPI-APs. Impairment of PGAP1 activity leads to developmental diseases in humans and fatality and infertility in animals. Here, we present three PGAP1 structures (2.66-2.84 Å), revealing its 10-transmembrane architecture and product-enzyme interaction details. PGAP1 holds GPI-AP acyl chains in an optimally organized, guitar-shaped cavity with apparent energetic penalties from hydrophobic-hydrophilic mismatches. However, abundant glycan-mediated interactions in the lumen counterbalance these repulsions, likely conferring substrate fidelity and preventing off-target hydrolysis of bulk membrane lipids. Structural and biochemical analyses uncover a serine hydrolase-type catalysis with atypical features and imply mechanisms for substrate entrance and product release involving a drawing compass movement of GPI-APs. Our findings advance the mechanistic understanding of GPI-AP remodeling.
Assuntos
Inositol , Proteínas de Membrana , Animais , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Inositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Hidrolases , Controle de Qualidade , Glicosilfosfatidilinositóis/químicaRESUMO
Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, has a poor prognosis and lacks effective treatment strategies. Here, the study discovered that TNBC shows a decreased expression of epithelial transcription factor ovo-like 2 (OVOL2). The loss of OVOL2 promotes fatty acid oxidation (FAO), providing additional energy and NADPH to sustain stemness characteristics, including sphere-forming capacity and tumor initiation. Mechanistically, OVOL2 not only suppressed STAT3 phosphorylation by directly inhibiting JAK transcription but also recruited histone deacetylase 1 (HDAC1) to STAT3, thereby reducing the transcriptional activation of downstream genes carnitine palmitoyltransferase1 (CPT1A and CPT1B). PyVT-Ovol2 knockout mice develop a higher number of primary breast tumors with accelerated growth and increased lung-metastases. Furthermore, treatment with FAO inhibitors effectively reduces stemness characteristics of tumor cells, breast tumor initiation, and metastasis, especially in OVOL2-deficient breast tumors. The findings suggest that targeting JAK/STAT3 pathway and FAO is a promising therapeutic strategy for OVOL2-deficient TNBC.
Assuntos
Ácidos Graxos , Oxirredução , Fator de Transcrição STAT3 , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Camundongos , Feminino , Ácidos Graxos/metabolismo , Humanos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Camundongos Knockout , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a signal peptide region that lacks consensus sequence and replaces it with GPI via a transamidation reaction. How GPI-T maintains broad specificity while preventing unintentional cleavage is unclear. Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity. In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements. Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft. GPI-T thus integrates various weak specificity regions to form strong selectivity and prevent accidental activation. These findings provide important mechanistic insights into GPI-anchored protein biogenesis.
Assuntos
Aminoaciltransferases , Glicosilfosfatidilinositóis , Humanos , Catálise , Domínio Catalítico , LigantesRESUMO
Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification by which poly ADP-ribose (PAR) polymers are covalently added to proteins through a PAR polymerase (PARP). Here, using proteomic approach, we identify the transcriptional regulator, OVOL2, is a novel substrate of PARP1 and can be PARylated at residues Lysine 145, Lysine 176, and Lysine 212 within its C2H2 zinc finger domains. Overexpression of PARylated OVOL2 alters cell morphology and induces lagging chromosomes and aneuploidy. To define the underlying molecular mechanism by which OVOL2 induces abnormal cell cycle and centrosome amplification, we uncover that the OVOL2 elevates the protein levels of Cyclin E by enhancing its stability. Furthermore, we identify Skp2, the E3 ubiquitin ligase of Cyclin E, as a direct target of PARylated OVOL2. Using ChIP assay, the OVOL2 binding site on the promoter region of Skp2 is mapped. To further explore the physiological effect, we show that PARylated OVOL2 can induce cell death. Furthermore, to investigate PARylated OVOL2 function in vivo, we further develop a null-mice xenograft model and generate MMTV-PyVT transgenic mice and monitor the effect of wild-type OVOL2 and non-PARylated OVOL2-3K/A mutants on tumor progression. Consistently, overexpression of wild-type OVOL2 in both null-mice xenograft and MMTV-PyVT transgenic mice displays significantly reduction of tumor progression, respectively, further indicating that the function of OVOL2 as a tumor suppressor in vivo is highly regulated by PARylation. Taken together, our study sheds new light on PARP1-induced PARylation as a critical event in the OVOL2-mediated regulation of chromosomal integrity and suppression of cancer cells growth.
Assuntos
Morte Celular/genética , Poli ADP Ribosilação/genética , Poli Adenosina Difosfato Ribose/genética , Fatores de Transcrição/genética , Aneuploidia , Animais , Sítios de Ligação/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Centrossomo/fisiologia , Cromossomos/genética , Ciclina E/genética , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Transgênicos , Poli(ADP-Ribose) Polimerase-1/genética , Regiões Promotoras Genéticas/genética , Proteômica/métodos , Proteínas Quinases Associadas a Fase S/genética , Transcrição Gênica/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Until now, two RNA demethylases have been identified, including FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homologue 5). As a mammalian m6A demethylase, ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles, and ALKBH5 may play a significant role in these biological processes. Nevertheless, no modulator of ALKBH5 has been reported. The reason for that may be the lack of in vitro assays for ALKBH5 inhibitor screening. Herein, we describe the development of two homogeneous assays for ALKBH5 using N6-methyladenosine as substrate with different principles. Using ALKBH5 recombinant, we developed a formaldehyde dehydrogenase coupled fluorescence based assay and an antibody based assay for the activity evaluation of ALKBH5. These robust coupled assays are suitable for screening ALKBH5 inhibitors in 384-well format (Z' factors of 0.74), facilitating the discovery of modulators in the quest for the regulation of biological processes.
Assuntos
Adenosina/análogos & derivados , Aldeído Oxirredutases/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Descoberta de Drogas/métodos , Imunofluorescência , Formaldeído/metabolismo , Ensaios de Triagem em Larga Escala/métodos , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Aldeído Oxirredutases/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Desmetilação , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Reprodutibilidade dos Testes , Espectrometria de FluorescênciaRESUMO
Wnt/ß-catenin signaling plays a key role in regulating adipogenesis through indirectly inhibiting the expression of C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ); however, the detailed molecular mechanism remains poorly understood. Moreover, the factor(s) that determines the Wnt/ß-catenin output level during adipogenesis is also not completely defined. In this study, we showed that Pygo2 exhibited a declined expression pattern during adipocyte differentiation, resulting in an attenuated Wnt/ß-catenin output level. The mechanism study indicated that Pygo2 inhibition led to the downregulation of Axin2, a constitutive Wnt target, in the cytoplasm. Consequently, Axin2-bound GSK3ß was released and translocated into the nucleus to phosphorylate C/EBPß and Snail, resulting in an increase in the DNA binding activity of C/EBPß and decreased protein stability of Snail, which subsequently activated the expression of C/EBPα and PPARγ. Consistent with this, embryonic fibroblasts from Pygo2-/- mice exhibited spontaneous adipocyte differentiation, and adipocyte precursor-specific Pygo2-deficient mice exhibited increased adiposity with decreased energy expenditure. We further showed impaired glucose tolerance and decreased systemic insulin sensitivity in Pygo2-deficient mice. Our study revealed an association between Pygo2 function and obesity or diabetes.
Assuntos
Adiposidade/genética , Glicemia/metabolismo , Homeostase/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Sinalização Wnt/fisiologia , Adipócitos/metabolismo , Adipogenia/genética , Tecido Adiposo Branco/metabolismo , Animais , Proteína Axina/metabolismo , Composição Corporal/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , beta Catenina/metabolismoRESUMO
Great progress has been achieved in the study of the role of TGF-ß signaling in triggering epithelial-mesenchymal transition (EMT) in a variety of cancers; however, the regulation of TGF-ß signaling during EMT in mammary tumor metastasis has not been completely defined. In the present study, we demonstrated that OVOL2, a zinc finger transcription factor, inhibits TGF-ß signaling-induced EMT in mouse and human mammary tumor cells, as well as in mouse tumor models. Data from the Oncomine databases indicated a strong negative relationship between OVOL2 expression and breast cancer progression. Moreover, our experiments revealed that OVOL2 inhibits TGF-ß signaling at multiple levels, including inhibiting Smad4 mRNA expression and inducing Smad7 mRNA expression, blocking the binding between Smad4 and target DNA, and interfering with complex formation between Smad4 and Smad2/3. These findings reveal a novel mechanism that controls the TGF-ß signaling output level in vitro and in vivo. The modulation of these molecular processes may represent a strategy for inhibiting breast cancer invasion by restoring OVOL2 expression.