Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 61
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
bioRxiv ; 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38746453

RESUMO

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIIIα/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease.

2.
Autophagy Rep ; 3(1)2024.
Artigo em Inglês | MEDLINE | ID: mdl-38344192

RESUMO

Macroautophagy (also known as autophagy) plays a pivotal role in maintaining cellular homeostasis. The terminal step of the multi-step autophagy degradation pathway involves fusion between the cargo-laden, double-membraned autophagosome and the lytic organelle lysosome/vacuole. Over the past decade, various core components of the molecular machinery that execute this critical terminal autophagy event have been identified. This review highlights recent advances in understanding the molecular structures, biochemical functions, and regulatory mechanisms of key components of this highly sophisticated machinery including the SNARE fusogens, tethering factors, Rab GTPases and associated guanine nucleotide exchange factors, and other accessory factors.

3.
Genes (Basel) ; 14(10)2023 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-37895210

RESUMO

Biallelic variants in the Golgi SNAP receptor complex member 2 gene (GOSR2) have been reported in progressive myoclonus epilepsy with neurodegeneration. Typical clinical features include ataxia and areflexia during early childhood, followed by seizures, scoliosis, dysarthria, and myoclonus. Here, we report two novel patients from unrelated families with a GOSR2-related disorder and novel genetic and clinical findings. The first patient, a male compound heterozygous for the GOSR2 splice site variant c.336+1G>A and the novel c.364G>A,p.Glu122Lys missense variant showed global developmental delay and seizures at the age of 2 years, followed by myoclonus at the age of 8 years with partial response to clonazepam. The second patient, a female homozygous for the GOSR2 founder variant p.Gly144Trp, showed only mild fine motor developmental delay and generalized tonic-clonic seizures triggered by infections during adolescence, with seizure remission on levetiracetam. The associated movement disorder progressed atypically slowly during adolescence compared to its usual speed, from initial intention tremor and myoclonus to ataxia, hyporeflexia, dysmetria, and dystonia. These findings expand the genotype-phenotype spectrum of GOSR2-related disorders and suggest that GOSR2 should be included in the consideration of monogenetic causes of dystonia, global developmental delay, and seizures.


Assuntos
Distonia , Distúrbios Distônicos , Epilepsias Mioclônicas Progressivas , Mioclonia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Ataxia/genética , Mutação , Epilepsias Mioclônicas Progressivas/genética , Proteínas Qb-SNARE/genética , Convulsões
4.
Elife ; 122023 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37417733

RESUMO

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.


Assuntos
Metabolismo dos Lipídeos , Transdução de Sinais , Regulação Alostérica , Transdução de Sinais/fisiologia , Fosforilação , Membrana Celular
5.
bioRxiv ; 2023 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-37090531

RESUMO

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCß activated by the IgE receptor, and Gßγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here using a combination of cryo electron microscopy, HDX-MS, and biochemical assays we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gßγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCß helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCß phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCß mediated phosphorylation. Overall, this works shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

6.
Cell Rep ; 42(3): 112172, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36842083

RESUMO

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.


Assuntos
Fosfatidilinositol 3-Quinases , Transdução de Sinais , Transdução de Sinais/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Acoplados a Proteínas G , Modelos Moleculares , Fosfatidilinositol 3-Quinase
7.
Mol Cell Biol ; 42(11): e0017022, 2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36190236

RESUMO

Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.


Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Nucleossomos/metabolismo , Regulação Alostérica , Proteínas de Saccharomyces cerevisiae/metabolismo , Histonas/metabolismo , Acetiltransferases/química , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Histona Acetiltransferases/metabolismo
8.
Genetics ; 222(3)2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36066422

RESUMO

The NuA4 lysine acetyltransferase complex acetylates histone and nonhistone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1-Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1Δ and epl1-CΔ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1Δ epl1-CΔ double mutants resembled the epl1-CΔ single mutants, indicating that Eaf1's role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionarily conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1-Eaf1 interaction regulated picNuA4 amount and activity.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Acetilação , Histonas/metabolismo , Fatores de Transcrição/metabolismo
9.
Biomedicines ; 10(9)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36140270

RESUMO

Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.

10.
Sci Rep ; 12(1): 12306, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35853968

RESUMO

Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to further structural investigations of SAGA. Here, we report a workflow for isolating/stabilizing yeast SAGA and preparing cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Microscopia Crioeletrônica , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
ACS Omega ; 7(4): 3424-3433, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35128251

RESUMO

Conserved from yeast to humans and composed of six core subunits (Elp1-Elp6), Elongator is a multiprotein complex that catalyzes the modification of the anticodon loop of transfer RNAs (tRNAs) and in turn regulates messenger RNA decoding efficiency. Previous studies showed that yeast Elongator consists of two subassemblies (yElp1/2/3 and yElp4/5/6) and adopts an asymmetric overall architecture. Yet, much less is known about the structural properties of the orthologous human Elongator. Furthermore, the order in which the different Elongator subunits come together to form the full assembly as well as how they coordinate with one another to catalyze tRNA modification is not fully understood. Here, we purified recombinant human Elongator subunits and subassemblies and examined them by single-particle electron microscopy. We found that the human Elongator complex is assembled from two subcomplexes that share similar overall morphologies as their yeast counterparts. Complementary co-purification and pulldown assays revealed that the scaffolding subunit human ELP1 (hELP1) has stabilizing effects on the human ELP3 catalytic subunit. Furthermore, the peripheral hELP2 subunit appears to enhance the integrity and substrate-binding ability of the dimeric hELP1/2/3. Lastly, we found that hELP4/5/6 is recruited to hELP1/2/3 via hELP3. Collectively, our work generated insights into the assembly process of core human Elongator and the coordination of different subunits within this complex.

12.
Structure ; 29(12): 1371-1381.e6, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34348129

RESUMO

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Anticorpos de Domínio Único/metabolismo , Animais , Domínio Catalítico/fisiologia , Humanos , Fosforilação
13.
Sci Adv ; 7(35)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34452907

RESUMO

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

14.
J Mol Biol ; 433(18): 167145, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34229011

RESUMO

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.


Assuntos
Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mamíferos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sítios de Ligação , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Mamíferos/genética , Conformação Proteica , Transporte Proteico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética
15.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
16.
Blood ; 137(26): 3641-3655, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33786587

RESUMO

The abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Sistemas de Liberação de Medicamentos , Leucemia Mieloide Aguda , Células-Tronco Neoplásicas/enzimologia , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Sulfonamidas/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase Axl
17.
Commun Biol ; 4(1): 291, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674710

RESUMO

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome-lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended "shepherd's staff" architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome-lysosome fusion. The hEPG5-GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5's overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Catarata/genética , Catarata/metabolismo , Predisposição Genética para Doença , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Proteólise , Células Sf9 , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/genética
18.
Commun Biol ; 3(1): 735, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277614

RESUMO

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Humanos , Insetos , Modelos Moleculares , Conformação Proteica , Isoformas de Proteínas , Especificidade por Substrato , Espectrometria de Massas em Tandem , Proteínas rab de Ligação ao GTP/genética
19.
Int J Mol Sci ; 21(21)2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33126766

RESUMO

Macroautophagy (also known as autophagy) is a major pathway for selective degradation of misfolded/aggregated proteins and damaged organelles and non-selective degradation of cytoplasmic constituents for the generation of power during nutrient deprivation. The multi-step degradation process, from sequestering cytoplasmic cargo into the double-membrane vesicle termed autophagosome to the delivery of the autophagosome to the lysosome or lytic vacuole for breakdown, is mediated by the core autophagy machinery composed of multiple Atg proteins, as well as the divergent sequence family of selective autophagy receptors. Single-particle electron microscopy (EM) is a molecular imaging approach that has become an increasingly important tool in the structural characterization of proteins and macromolecular complexes. This article summarizes the contributions single-particle EM have made in advancing our understanding of the core autophagy machinery and selective autophagy receptors. We also discuss current technical challenges and roadblocks, as well as look into the future of single-particle EM in autophagy research.


Assuntos
Autofagossomos , Autofagia , Microscopia Eletrônica/métodos , Imagem Individual de Molécula/métodos , Animais , Humanos , Vacúolos
20.
Sci Rep ; 8(1): 17353, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478341

RESUMO

Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped viruses (e.g. hepatitis A virus) can acquire an envelope during infection by hijacking host cellular pathways. In this study, we examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket paralysis virus (CrPV). Utilizing quantitative proteomics, we demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the diverse strategies of nonenveloped viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission.


Assuntos
Dicistroviridae/patogenicidade , Drosophila melanogaster/virologia , Vesículas Extracelulares/virologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas Virais/metabolismo , Animais , Dicistroviridae/genética , Dicistroviridae/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Exossomos/virologia , RNA Viral/metabolismo , Proteínas Virais/análise
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...