RESUMEN
S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes. However, it has been suggested that changes in the total level of CDK kinase activity, rather than substrate specificity, drive the temporal ordering of S phase and mitosis. Here, we present a phosphoproteomics-based systems analysis of CDK substrates in fission yeast and demonstrate that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK. This is achieved by rising CDK activity and the differential sensitivity of substrates to CDK activity over a wide dynamic range. This is combined with rapid phosphorylation turnover to generate clearly resolved substrate-specific activity thresholds, which in turn ensures the appropriate ordering of downstream cell-cycle events. Comparative analysis with wild-type cells expressing multiple cyclin-CDK complexes reveals how cyclin-substrate specificity works alongside activity thresholds to fine-tune the patterns of substrate phosphorylation.
Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Ciclinas/metabolismo , Mitosis , FosforilaciónRESUMEN
Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
Asunto(s)
Proteínas de Ciclo Celular/ultraestructura , Cromátides/ultraestructura , Proteínas Cromosómicas no Histona/ultraestructura , ADN/ultraestructura , Intercambio de Cromátides Hermanas/genética , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Microscopía por Crioelectrón , ADN/genética , Conformación de Ácido Nucleico , Conformación Proteica , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , CohesinasRESUMEN
The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico , GMP Cíclico , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Fosforilación , GMP Cíclico/metabolismo , Malaria/parasitología , Malaria/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Animales , Plasmodium falciparum/metabolismo , Plasmodium falciparum/genética , Humanos , Transducción de Señal , Eritrocitos/parasitología , Eritrocitos/metabolismoRESUMEN
DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.
Asunto(s)
Carcinogénesis/patología , ADN Polimerasa II/química , ADN Polimerasa II/fisiología , Replicación del ADN , Discapacidades del Desarrollo/etiología , Trastornos del Crecimiento/etiología , Leucopenia/etiología , Animales , Carcinogénesis/genética , Células Cultivadas , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Femenino , Humanos , Recién Nacido , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.
Asunto(s)
Macrófagos/metabolismo , Fagosomas/metabolismo , Animales , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Fosforilación/fisiología , Proteínas/metabolismo , Transducción de Señal/fisiología , Staphylococcus aureus/metabolismoRESUMEN
The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Fosfoserina/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN/biosíntesis , ADN/química , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , Fibroblastos , Genoma/genética , Células HEK293 , Humanos , Ratones , Mutación , Fenotipo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fase S , Complejo Shelterina , Telomerasa/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-kB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138Aalteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-kB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1, which stimulated keratinocyte metabolism, but not for NF-kB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.
RESUMEN
The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Histona Demetilasas con Dominio de Jumonji , Proteínas de Ciclo Celular/metabolismo , Humanos , Hidroxilación , Proteínas Intrínsecamente Desordenadas/metabolismo , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Dominios Proteicos , Factores de Transcripción/metabolismoRESUMEN
Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.
Asunto(s)
Toxoplasma , Toxoplasma/metabolismo , Calcio/metabolismo , Nucleótidos Cíclicos/metabolismo , Retroalimentación , LípidosRESUMEN
Loss-of-function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early-onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule-associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3-labelled plus-end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient-derived human neurons. Our results reveal a novel activity-dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder.
Asunto(s)
Dendritas/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Síndromes Epilépticos/genética , Síndromes Epilépticos/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Espasmos Infantiles/genética , Espasmos Infantiles/metabolismoRESUMEN
Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACß) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.
Asunto(s)
AMP Cíclico/metabolismo , Interacciones Huésped-Parásitos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Parásitos/metabolismo , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Parásitos/enzimología , Parásitos/crecimiento & desarrollo , Parásitos/ultraestructura , Fosfoproteínas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/patogenicidad , Plasmodium falciparum/ultraestructura , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite.
Asunto(s)
Malaria Falciparum/genética , Plasmodium falciparum/genética , Serina Proteasas/química , Subtilisina/genética , Secuencia de Aminoácidos/genética , Animales , Eritrocitos/parasitología , Genoma/genética , Humanos , Estadios del Ciclo de Vida/genética , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Plasmodium falciparum/patogenicidad , Proteolisis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Serina Proteasas/genética , Subtilisina/química , Vacuolas/parasitologíaRESUMEN
Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-ß-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8(D270A/D270A) mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8(D270A) mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.
Asunto(s)
Macrófagos/enzimología , Proteínas Quinasas/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Toll-Like/metabolismo , Animales , Activación Enzimática , Espectrometría de Masas , RatonesRESUMEN
Four-way DNA intermediates, also known as Holliday junctions, are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. Here we identify nucleases from Saccharomyces cerevisiae and human cells that promote Holliday junction resolution, in a manner analogous to that shown by the Escherichia coli Holliday junction resolvase RuvC. The human Holliday junction resolvase, GEN1, and its yeast orthologue, Yen1, were independently identified using two distinct experimental approaches: GEN1 was identified by mass spectrometry following extensive fractionation of HeLa cell-free extracts, whereas Yen1 was detected by screening a yeast gene fusion library for nucleases capable of Holliday junction resolution. The eukaryotic Holliday junction resolvases represent a new subclass of the Rad2/XPG family of nucleases. Recombinant GEN1 and Yen1 resolve Holliday junctions by the introduction of symmetrically related cuts across the junction point, to produce nicked duplex products in which the nicks can be readily ligated.
Asunto(s)
Resolvasas de Unión Holliday/aislamiento & purificación , Resolvasas de Unión Holliday/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ADN/química , ADN/metabolismo , Reparación del ADN , Células HeLa , Resolvasas de Unión Holliday/química , Resolvasas de Unión Holliday/genética , Humanos , Recombinación Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Protein glycosylation, a complex and heterogeneous post-translational modification that is frequently dysregulated in disease, has been difficult to analyse at scale. Here we report a data-independent acquisition technique for the large-scale mass-spectrometric quantification of glycopeptides in plasma samples. The technique, which we named 'OxoScan-MS', identifies oxonium ions as glycopeptide fragments and exploits a sliding-quadrupole dimension to generate comprehensive and untargeted oxonium ion maps of precursor masses assigned to fragment ions from non-enriched plasma samples. By applying OxoScan-MS to quantify 1,002 glycopeptide features in the plasma glycoproteomes from patients with COVID-19 and healthy controls, we found that severe COVID-19 induces differential glycosylation in IgA, haptoglobin, transferrin and other disease-relevant plasma glycoproteins. OxoScan-MS may allow for the quantitative mapping of glycoproteomes at the scale of hundreds to thousands of samples.
Asunto(s)
COVID-19 , Glicopéptidos , Humanos , Espectrometría de Masas , Glicosilación , Glicopéptidos/análisis , Glicopéptidos/química , Glicopéptidos/metabolismo , IonesRESUMEN
A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins, the Traf and Nck interacting kinase (TNIK), is a postsynaptic density (PSD) signaling hub, with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment, the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here, using hiPSC-derived excitatory neurons, we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover, the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses, suggesting a differential role of TNIK in immature synapsis in NDD.
RESUMEN
The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However - in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins.
Asunto(s)
Anticuerpos Antiprotozoarios/farmacología , Antígenos de Protozoos/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Recombinación Homóloga , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases, implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models, we show that only dual loss of Ndr1/2 in neurons causes neurodegeneration. This phenotype was present when NDR kinases were deleted both during embryonic development, as well as in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein Raph1/Lpd1, as a novel NDR1/2 substrate, and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking, and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health.
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Autofagia , Proteómica , Femenino , Embarazo , Animales , Ratones , Autofagosomas , Neuronas , Proteostasis , Proteínas de la Membrana/genética , MamíferosRESUMEN
PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.
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Transporte de ARN , ARN , Humanos , Transporte Activo de Núcleo Celular , Expresión Génica , HomeostasisRESUMEN
The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important merozoite surface and PV proteins. These include the essential merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptry-associated protein 1) and the merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.