RESUMEN
The increasing implication of lymphocytes in general physiology and immune surveillance outside of infection poses the question of how their antigen receptors might be involved. Here, we show that macromolecular aggregates of intraepidermal γδ T cell antigen receptors (TCRs) in the mouse skin aligned with and depended on Skint1, a butyrophilin-like (BTNL) protein expressed by differentiated keratinocytes (KCs) at steady state. Interruption of TCR-mediated 'normality sensing' had no impact on γδ T cell numbers but altered their signature phenotype, while the epidermal barrier function was compromised. In addition to the regulation of steady-state physiology, normality sensing licensed intraepidermal T cells to respond rapidly to subsequent tissue perturbation by using innate tumor necrosis factor (TNF) superfamily receptors. Thus, interfering with Skint1-dependent interactions between local γδ T cells and KCs at steady state increased the susceptibility to ultraviolet B radiation (UVR)-induced DNA damage and inflammation, two cancer-disposing factors.
Asunto(s)
Linfocitos Intraepiteliales , Receptores de Antígenos de Linfocitos T gamma-delta , Animales , Butirofilinas , Epidermis , Linfocitos Intraepiteliales/metabolismo , Concesión de Licencias , Ratones , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismoRESUMEN
Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.
Asunto(s)
Metiltransferasas/metabolismo , ARN Mitocondrial/química , ARN de Transferencia/química , Animales , Anticodón , Proliferación Celular , Codón , Citoplasma , ADN Mitocondrial/metabolismo , Transporte de Electrón , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Membranas Mitocondriales , Proteínas Mitocondriales/química , Consumo de Oxígeno , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Ribosomas/metabolismo , Regulación hacia ArribaRESUMEN
Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.
Asunto(s)
Ferroptosis , Vitamina K , Antídotos/farmacología , Antioxidantes/metabolismo , Antioxidantes/farmacología , Ligasas de Carbono-Carbono/metabolismo , Coenzimas/metabolismo , Ferroptosis/efectos de los fármacos , Hidroquinonas/metabolismo , Hidroquinonas/farmacología , Peroxidación de Lípido/efectos de los fármacos , Oxidación-Reducción , Proteína de Unión al Calcio S100A4/metabolismo , Vitamina K/metabolismo , Vitamina K/farmacología , Warfarina/efectos adversosRESUMEN
Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Animales , Humanos , Ratones , Persona de Mediana Edad , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Disfunción Cognitiva , Ratones Transgénicos , Placa Amiloide/metabolismo , Proteínas tau/metabolismoRESUMEN
Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.
Asunto(s)
Glucemia/metabolismo , Antagonistas de Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Animales , Glucemia/análisis , Línea Celular , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula , Clatrina/metabolismo , Células Endocrinas/metabolismo , Endocitosis , Retículo Endoplásmico/metabolismo , Prueba de Tolerancia a la Glucosa , Aparato de Golgi/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Proteínas de Neoplasias/química , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacologíaRESUMEN
The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.
Asunto(s)
Proteína A Centromérica , Centrómero , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/química , Humanos , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Unión Proteica , Ciclo Celular/genética , Modelos Moleculares , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Nucleosomas/metabolismo , Nucleosomas/química , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.
Asunto(s)
Autoinmunidad , Encefalomielitis Autoinmune Experimental , Ratones , Animales , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Inflamación/metabolismo , Diferenciación Celular , Encefalomielitis Autoinmune Experimental/genética , Receptores de Antígenos de Linfocitos T/genética , Ubiquitina-Proteína LigasasRESUMEN
The p14ARF protein is a well-known regulator of p53-dependent and p53-independent tumor-suppressive activities. In unstressed cells, p14ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14ARF undergoes an immediate redistribution to the nucleo- and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C-terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14ARF . In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14ARF . Genotoxic stress causes augmented interaction between PRMT1 and p14ARF , accompanied by arginine methylation of p14ARF . PRMT1-dependent NLS/NoLS methylation promotes the release of p14ARF from NPM and nucleolar sequestration, subsequently leading to p53-independent apoptosis. This PRMT1-p14ARF cooperation is cancer-relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1-mediated arginine methylation is an important trigger for p14ARF 's stress-induced tumor-suppressive function.
Asunto(s)
Neoplasias Pancreáticas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Ciclo Celular/fisiología , Línea Celular , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , Células Sf9 , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias PancreáticasRESUMEN
The BAFF/APRIL-system with the two cytokines BAFF and APRIL and their three receptors, transmembrane activator and CAML interactor (TACI), BAFF receptor, and B-cell maturation Ag, is important for B cell maintenance. The BAFF/APRIL system is a therapeutic target in B cell-derived malignancies and autoimmune diseases. However, unexpected outcomes of clinical trials with atacicept (TACI-Fc) underline our incomplete understanding of this system. Shedding of the three receptors is one important regulatory element. In humans, TACI exists in two isoforms generated through alternative splicing in their extracellular portion: TACI-long (l) has two cysteine-rich domains, whereas TACI-short (s) lacks the first low-affinity one. In this study, we discriminated soluble (s) forms of TACI-l and TACI-s with newly generated mAbs and found that both were spontaneously released from activated human B cells, with a predominance of sTACI-l. Furthermore, sTACI-l was also the dominant isoform in human serum. Vaccination with the mRNA vaccine from BioNTech does not significantly affect the serum levels of sTACI-l. Both TACI-s and TACI-l were shed by a disintegrin and metalloproteinase domain-containing protein 10. TACI-l and TACI-s formed homo- and hetero-oligomers in soluble and membrane-bound forms. Both sTACI-l and sTACI-s acted as decoy receptors for BAFF, but only sTACI-l also efficiently inhibited APRIL. Dimerization of sTACI-l enhanced its decoy functions only slightly. Together, we extend our knowledge of the complexity of the BAFF/APRIL system by identifying and characterizing the two soluble isoforms of TACI.
Asunto(s)
Linfocitos B , Proteína Activadora Transmembrana y Interactiva del CAML , Humanos , Empalme Alternativo , Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/genética , Citocinas/genética , Isoformas de Proteínas/genética , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.
Asunto(s)
Centrosoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/embriología , Microtúbulos/metabolismo , Neurogénesis , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Humanos , Uniones Intercelulares/metabolismo , Interfase , Ventrículos Laterales/anatomía & histología , Glándulas Mamarias Animales/citología , Ratones , Tamaño de los Órganos , Organoides/citologíaRESUMEN
The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.
Asunto(s)
Proteínas de Unión al ARN , Factores de Transcripción , Humanos , Proteínas de Unión al ADN/genética , Epilepsia , Cuerpos de Procesamiento , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Repair of covalent DNA-protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina/fisiología , Ubiquitinación , Catálisis , Línea Celular , Cromatina/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/química , Enzimas Desubicuitinizantes/metabolismo , Técnicas de Inactivación de Genes , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Peptidasa Específica de Ubiquitina 7/fisiologíaRESUMEN
Proton (H+) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few proton channels have been identified so far. Here, we identify a family of voltage-gated "pacemaker" channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpolarization and conducts protons into the cytosol. Surprisingly, protons permeate through the channel's voltage-sensing domain, whereas the pore domain is nonfunctional. Key to proton permeation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional domains, HCNL1 evolutionarily adapted to a low-Na+ freshwater environment to conserve sperm's ability to depolarize.
Asunto(s)
Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Masculino , Familia de Multigenes , Protones , Espermatozoides/metabolismo , Pez Cebra/genéticaRESUMEN
Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.
Asunto(s)
Envejecimiento/metabolismo , Amiloide/metabolismo , Antígenos de Superficie/metabolismo , Proteínas de la Leche/metabolismo , Enfermedades Vasculares/metabolismo , Anciano de 80 o más Años , Amiloide/genética , Animales , Antígenos de Superficie/genética , Aorta/metabolismo , Aorta/patología , Química Encefálica/fisiología , Circulación Cerebrovascular/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de la Leche/genética , Enfermedades Vasculares/patologíaRESUMEN
Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes. Protein-protein interaction assays revealed the in vivo formation of MDL1, MDL2, and MDL3 homo-oligomers, as well as the formation of MDL1-MDL2 hetero-oligomers. Functionally, Arabidopsismdl mutants exhibited a delayed transition from vegetative to reproductive growth (flowering) under long-day conditions, but not in a short-day environment. In addition, mdl mutants were more resistant to colonization by the bacterial pathogen Pseudomonas syringae pv. maculicola. The latter phenotype was compromised by the additional mutation of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), a gene implicated in the defense-induced biosynthesis of the key signaling molecule salicylic acid. However, the enhanced antibacterial immunity was not associated with any constitutive or pathogen-induced alterations in the levels of characteristic phytohormones or defense-associated metabolites. Interestingly, bacterial infection triggered relocalization and accumulation of MDL1 and MDL2 at the peripheral lobes of leaf epidermal cells. Collectively, our data indicate redundant functionality and a complex interplay between the three chemokine-like Arabidopsis MDL proteins in the regulation of both developmental and immune-related processes. These insights expand the comparative cross-kingdom analysis of MIF/MDL signaling in human and plant systems.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Quimiocinas/metabolismo , Flores/inmunología , Inmunidad Innata/inmunología , Enfermedades de las Plantas/inmunología , Pseudomonas syringae/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Flores/microbiología , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/microbiologíaRESUMEN
Death receptor 6 (DR6) is an orphan member of the TNF receptor superfamily and controls cell death and differentiation in a cell-autonomous manner in different cell types. Here, we report an additional non-cell-autonomous function for DR6 in the peripheral nervous system (PNS). DR6-knockout (DR6 KO) mice showed precocious myelination in the PNS Using an in vitro myelination assay, we demonstrate that neuronal DR6 acts in trans on Schwann cells (SCs) and reduces SC proliferation and myelination independently of its cytoplasmic death domain. Mechanistically, DR6 was found to be cleaved in neurons by "a disintegrin and metalloprotease 10" (ADAM10), releasing the soluble DR6 ectodomain (sDR6). Notably, in the in vitro myelination assay, sDR6 was sufficient to rescue the DR6 KO phenotype. Thus, in addition to the cell-autonomous receptor function of full-length DR6, the proteolytically released sDR6 can unexpectedly also act as a paracrine signaling factor in the PNS in a non-cell-autonomous manner during SC proliferation and myelination. This new mode of DR6 signaling will be relevant in future attempts to target DR6 in disease settings.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proliferación Celular , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Células de Schwann/metabolismo , Animales , Muerte Celular , Línea Celular , Citoplasma/metabolismo , Dominio de Muerte , Desintegrinas/metabolismo , Femenino , Células HEK293 , Humanos , Hibridomas , Masculino , Metaloproteasas/metabolismo , Ratones , Ratones Noqueados , Vaina de Mielina/metabolismo , Comunicación Paracrina , Fenotipo , Receptores del Factor de Necrosis Tumoral/genética , Células de Schwann/ultraestructura , Especificidad por SustratoRESUMEN
Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m6A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m1A, m5C, m7G, 2'-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.
Asunto(s)
Anticuerpos/genética , Ribonucleótidos/genética , Nucleótidos/genética , ARN/genética , ARN Mensajero/genéticaRESUMEN
Cryptochromes are photolyase-related blue-light receptors acting as core components of the mammalian circadian clock in the cell nuclei. One or more members of the cryptochrome protein family are also assumed to play a role in avian magnetoreception, but the primary sensory molecule in the retina of migratory birds that mediates light-dependent magnetic compass orientation has still not been identified. The mRNA of cryptochrome 2 (Cry2) has been reported to be located in the cell nuclei of the retina, but Cry2 localisation has not yet been demonstrated at the protein level. Here, we provide evidence that Cry2 protein is located in the photoreceptor inner segments, the outer nuclear layer, the inner nuclear layer and the ganglion cell layer in the retina of night-migratory European robins, homing pigeons and domestic chickens. At the subcellular level, we find Cry2 both in the cytoplasm and the nucleus of cells residing in these layers. This broad nucleic expression rather points to a role for avian Cry2 in the circadian clock and is consistent with a function as a transcription factor, analogous to mammalian Cry2, and speaks against an involvement in magnetoreception.
Asunto(s)
Criptocromos , Pájaros Cantores , Animales , Pollos , Criptocromos/metabolismo , Mamíferos/metabolismo , Retina/fisiología , Pájaros Cantores/fisiología , Factores de Transcripción/metabolismoRESUMEN
Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)-interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19-cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/metabolismo , Biología Computacional/métodos , Ciclina C/genética , Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasa 8 Dependiente de Ciclina/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitosis , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Abnormal generation of neurotoxic amyloid-ß peptide (Aß) 42/43 species due to mutations in the catalytic presenilin 1 (PS1) subunit of γ-secretase is the major cause of familial Alzheimer's disease (FAD). Deeper mechanistic insight on the generation of Aß43 is still lacking, and it is unclear whether γ-secretase modulators (GSMs) can reduce the levels of this Aß species. By comparing several types of Aß43-generating FAD mutants, we observe that very high levels of Aß43 are often produced when presenilin function is severely impaired. Altered interactions of C99, the precursor of Aß, are found for all mutants and are independent of their particular effect on Aß production. Furthermore, unlike previously described GSMs, the novel compound RO7019009 can effectively lower Aß43 production of all mutants. Finally, substrate-binding competition experiments suggest that RO7019009 acts mechanistically after initial C99 binding. We conclude that altered C99 interactions are a common feature of diverse types of PS1 FAD mutants and that also patients with Aß43-generating FAD mutations could in principle be treated by GSMs.