RESUMO
In nonalcoholic fatty liver disease (NAFLD) the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 variant is a contributor. In mice, the Pnpla3 148M variant accumulates on lipid droplets and probably leads to sequestration of a lipase cofactor leading to impaired mobilization of triglycerides. To advance our understanding of the localization and abundance of PNPLA3 protein in humans, we used liver biopsies from patients with NAFLD to investigate the link to NAFLD and the PNPLA3 148M genotype. We experimentally qualified an antibody against human PNPLA3. Hepatic PNPLA3 protein fractional area and localization were determined by immunohistochemistry in biopsies from a well-characterized NAFLD cohort of 67 patients. Potential differences in hepatic PNPLA3 protein levels among patients related to degree of steatosis, lobular inflammation, ballooning, and fibrosis, and PNPLA3 I148M gene variants were assessed. Immunohistochemistry staining in biopsies from patients with NAFLD showed that hepatic PNPLA3 protein was predominantly localized to the membranes of small and large lipid droplets in hepatocytes. PNPLA3 protein levels correlated strongly with steatosis grade (p = 0.000027) and were also significantly higher in patients with lobular inflammation (p = 0.009), ballooning (p = 0.022), and significant fibrosis (stage 2-4, p = 0.014). In addition, PNPLA3 levels were higher in PNPLA3 rs738409 148M (CG, GG) risk allele carriers compared to 148I (CC) nonrisk allele carriers (p = 0.0029). Conclusion: PNPLA3 protein levels were associated with increased hepatic lipid content and disease severity in patients with NAFLD and were higher in PNPLA3 rs738409 (148M) risk allele carriers. Our hypothesis that increased hepatic levels of PNPLA3 may be part of the pathophysiological mechanism of NAFLD is supported.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Aciltransferases , Alelos , Animais , Fibrose , Humanos , Inflamação/genética , Lipase/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Fosfolipases/genética , Fosfolipases A2 Independentes de Cálcio/genética , TriglicerídeosRESUMO
Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.
Assuntos
Suscetibilidade a Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Alelos , Animais , Biomarcadores , Linhagem Celular , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica , Variação Genética , Genótipo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Polimorfismo de Nucleotídeo Único , RNA-Seq , RibonucleasesRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.
Assuntos
Anticorpos/imunologia , Especificidade de Anticorpos , Antígeno B7-H1/imunologia , Sítios de Ligação de Anticorpos , Mapeamento de Epitopos , Imuno-Histoquímica , Neoplasias/imunologia , Anticorpos/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Glicosilação , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Valor Preditivo dos Testes , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Esquizofrenia/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Bioensaio , Humanos , Estrutura MolecularRESUMO
Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.
Assuntos
Proteínas de Drosophila/genética , Complexo Dinactina/genética , Dineínas/genética , Animais , Sítios de Ligação , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Complexo Dinactina/química , Dineínas/química , Ligação Proteica/genética , Multimerização Proteica , Transporte Proteico/genética , Transporte de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genéticaRESUMO
The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica , Multimerização ProteicaRESUMO
Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies.
Assuntos
Centrossomo/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Mitose , Animais , Sítios de Ligação , Centríolos/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. Here we describe a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end-directed mRNA transport during Drosophila development. We identified the protein Lissencephaly-1 (Lis1) and found that minus-end travel distances of localizing transcripts are dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, we uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, we provide evidence that Lis1 levels regulate the overall association of dynein with dynactin. Our data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , Animais , Complexo Dinactina , Transporte Proteico , Transporte de RNARESUMO
BACKGROUND: centrosomes are major microtubule organizing centers in animal cells, and they comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Centrosome size is tightly regulated during the cell cycle, and it has recently been shown that the two centrosomes in certain stem cells are often asymmetric in size. There is compelling evidence that centrioles influence centrosome size, but how centrosome size is set remains mysterious. RESULTS: we show that the conserved Drosophila PCM protein Cnn exhibits an unusual dynamic behavior, because Cnn molecules only incorporate into the PCM closest to the centrioles and then spread outward through the rest of the PCM. Cnn incorporation into the PCM is driven by an interaction with the conserved centriolar proteins Asl (Cep152 in humans) and DSpd-2 (Cep192 in humans). The rate of Cnn incorporation into the PCM is tightly regulated during the cell cycle, and this rate influences the amount of Cnn in the PCM, which in turn is an important determinant of overall centrosome size. Intriguingly, daughter centrioles in syncytial embryos only start to incorporate Cnn as they disengage from their mothers; this generates a centrosome size asymmetry, with mother centrioles always initially organizing more Cnn than their daughters. CONCLUSIONS: centrioles can control the amount of PCM they organize by regulating the rate of Cnn incorporation into the PCM. This mechanism can explain how centrosome size is regulated during the cell cycle and also allows mother and daughter centrioles to set centrosome size independently of one another.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Proteínas de Homeodomínio/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Proteínas de Homeodomínio/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In C. elegans, genome-wide screens have identified just five essential centriole-duplication factors: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 [1-8]. These proteins are widely believed to comprise a conserved core duplication module [3, 9-14]. In worm embryos, SPD-2 is the most upstream component of this module, and it is also essential for pericentriolar material (PCM) recruitment to the centrioles [1, 4, 15, 16]. Here, we show that Drosophila Spd-2 (DSpd-2) is a component of both the centrioles and the PCM and has a role in recruiting PCM to the centrioles. DSpd-2 appears not, however, to be essential for centriole duplication in somatic cells. Moreover, PCM recruitment in DSpd-2 mutant somatic cells is only partially compromised, and mitosis appears unperturbed. In contrast, DSpd-2 is essential for proper PCM recruitment to the fertilizing sperm centriole, and hence for microtubule nucleation and pronuclear fusion. DSpd-2 therefore appears to have a particularly important role in recruiting PCM to the sperm centriole. We speculate that the SPD-2 family of proteins might only be absolutely essential for the recruitment of centriole duplication factors and PCM to the centriole(s) that enter the egg with the fertilizing sperm.