RESUMEN
Calcium phosphate nanoparticles (100 nm) were fluorescently labelled with poly(ethyleneimine) (PEIATTO490LS; red fluorescence). They were loaded with a Tandem fusion protein consisting of mRFP1-eGFP (red and green fluorescence in the same molecule)that acts as smart biological pH sensor to trace nanoparticles inside cells. Its fluorescence is also coupled to the structural integrity of the protein, i.e. it is also a label for a successful delivery of a functional protein into the cell. At pH 7.4, the fluorescence of both proteins (red and green) is detectable. At a pH of 4.5-5 inside the lysosomes, the green fluorescence is quenched due to the protonation of the eGFP chromophore, but the pH-independent red fluorescence of mRFP1 remains. The nanoparticles were taken up by cells (cell lines: HeLa, Caco-2 and A549) via endocytic pathways and then directed to lysosomes. Time-resolved confocal laser scanning microscopy confirmed mRFP1 and nanoparticles co-localizing with lysosomes. The fluorescence of eGFP was only detectable outside lysosomes, i.e. most likely inside early endosomes or at the cell membrane during the uptake, indicating the neutral pH at these locations. The Tandem fusion protein provides a versatile platform to follow the intracellular pathway of bioactive nanocarriers, e.g. therapeutic proteins. The transfection with a Tandem-encoding plasmid by calcium phosphate nanoparticles led to an even intracellular protein distribution in cytosol and nucleoplasm, i.e. very different from direct protein uptake. Neither dissolved protein nor dissolved plasmid DNA were taken up by the cells, underscoring the necessity for a suitable carrier like a nanoparticle. STATEMENT OF SIGNIFICANCE: A pH-sensitive protein ("tandem") was used to follow the pathway of calcium phosphate nanoparticles. This protein consists of a pH-sensitive fluorophore (eGFP; green) and a pH-independent fluorophore (mRFP1; red). This permits to follow the pathway of a nanoparticle inside a cell. At a low pH inside an endolysosome, the green fluorescence vanishes but the red fluorescence persists. This is also a very useful model for the delivery of therapeutic proteins into cells. The delivery by nanoparticles was compared with the protein expression after cell transfection with plasmid DNA encoding for the tandem protein. High-resolution image analysis gave quantitative data on the intracellular protein distribution.
Asunto(s)
Nanopartículas , Células CACO-2 , Fosfatos de Calcio , Proteínas Fluorescentes Verdes/genética , Humanos , Concentración de Iones de Hidrógeno , TransfecciónRESUMEN
Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Omura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Productos Biológicos/farmacología , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Productos Biológicos/química , Estructura MolecularRESUMEN
Lysosomal membrane permeabilization or full rupture of lysosomes is a common and severe stress condition that is relevant for degenerative disease, infection and cancer. If damage is limited, cells can repair lysosomes by means of the endosomal sorting complex required for transport (ESCRT) machinery. Presumably, if repair fails, lysosomes are tagged with ubiquitin to initiate clearance by selective macroautophagy, termed lysophagy. Accumulating evidence suggests damage-induced exposure of luminal glycans to the cytosol as the key trigger for ubiquitination. In this review, we discuss recent data on cellular damage sensing, the underlying ubiquitination and autophagy machinery as well as additional layers of regulation such as processing of ubiquitinated proteins by the AAA-ATPase VCP/p97. We conclude with thoughts on how these mechanisms may regulate decision making between lysosome repair and lysophagy.
Asunto(s)
Lisosomas/metabolismo , Macroautofagia , Animales , Humanos , Permeabilidad , Ubiquitina/metabolismo , Ubiquitinación , Proteína que Contiene Valosina/metabolismoRESUMEN
The autophagic clearance of damaged lysosomes by lysophagy involves extensive modification of the organelle with ubiquitin, but the underlying ubiquitination machinery is still poorly characterized. Here, we use an siRNA screening approach and identify human UBE2QL1 as a major regulator of lysosomal ubiquitination, lysophagy, and cell survival after lysosomal damage. UBE2QL1 translocates to permeabilized lysosomes where it associates with damage sensors, ubiquitination targets, and lysophagy effectors. UBE2QL1 knockdown reduces ubiquitination and accumulation of the critical autophagy receptor p62 and abrogates recruitment of the AAA-ATPase VCP/p97, which is essential for efficient lysophagy. Crucially, it affects association of LC3B with damaged lysosomes indicating that autophagosome formation was impaired. Already in unchallenged cells, depletion of UBE2QL1 leads to increased lysosomal damage, mTOR dissociation from lysosomes, and TFEB activation pointing to a role in lysosomal homeostasis. In line with this, mutation of the homologue ubc-25 in Caenorhabditis elegans exacerbates lysosome permeability in worms lacking the lysosome stabilizing protein SCAV-3/LIMP2. Thus, UBE2QL1 coordinates critical steps in the acute endolysosomal damage response and is essential for maintenance of lysosomal integrity.
Asunto(s)
Autofagia , Endosomas/metabolismo , Lisosomas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Adenosina Trifosfatasas , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Supervivencia Celular , Endosomas/ultraestructura , Galectinas/metabolismo , Células HeLa , Humanos , Lisina/metabolismo , Lisosomas/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares , Permeabilidad , ARN Interferente Pequeño/metabolismo , Proteína Sequestosoma-1/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
Differentiated tissue is particularly vulnerable to alterations in protein and organelle homeostasis. The essential protein VCP, mutated in hereditary inclusion body myopathy, amyotrophic lateral sclerosis and frontotemporal dementia, is critical for efficient clearance of misfolded proteins and damaged organelles in dividing cells, but its role in terminally differentiated tissue affected by disease mutations is less clear. To understand the relevance of VCP in differentiated tissue, we inactivated it in skeletal muscle of adult mice. Surprisingly, knockout muscle demonstrated a necrotic myopathy with increased macroautophagic/autophagic proteins and damaged lysosomes. This was not solely due to a defect in autophagic degradation because age-matched mice with muscle inactivation of the autophagy essential protein, ATG5, did not demonstrate a myopathy. Notably, myofiber necrosis was preceded by upregulation of LGALS3/Galectin-3, a marker of damaged lysosomes, and TFEB activation, suggesting early defects in the lysosomal system. Consistent with that, myofiber necrosis was recapitulated by chemical induction of lysosomal membrane permeabilization (LMP) in skeletal muscle. Moreover, TFEB was activated after LMP in cells, but activation and nuclear localization of TFEB persisted upon VCP inactivation or disease mutant expression. Our data identifies VCP as central mediator of both lysosomal clearance and biogenesis in skeletal muscle. Abbreviations: AAA: ATPases Associated with diverse cellular Activities; TUBA1A/α-tubulin: tubulin alpha 1a; ATG5: autophagy related 5; ATG7: autophagy related 7; ACTA1: actin alpha 1, skeletal muscle; CLEAR: coordinated lysosomal expression and regulation; CTSB/D: cathepsin B/D; Ctrl: control; DAPI: diamidino-2-phenylindole; EBSS: Earle's balanced salt solution; ELDR: endolysosomal damage response; ESCRT: endosomal sorting complexes required for transport; Gastroc/G: gastrocnemius; H&E: hematoxylin and eosin; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; IBMPFD/ALS: inclusion body myopathy associated with Paget disease of the bone, frontotemporal dementia and amyotrophic lateral sclerosis; i.p.: intraperitoneal; LAMP1/2: lysosomal-associated membrane protein 1/2; LLOMe: Leu-Leu methyl ester hydrobromide; LGALS3/Gal3: galectin 3; LMP: lysosomal membrane permeabilization; MTOR: mechanistic target of rapamycin kinase; MYL1: myosin light chain 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MSP: multisystem proteinopathy; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; Quad/Q: quadriceps; RHEB: Ras homolog, mTORC1 binding; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TA: tibialis anterior; siRNA: small interfering RNA; SQSTM1/p62, sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBS: Tris-buffered saline; TXFN, tamoxifen; UBXN6/UBXD1: UBX domain protein 6; VCP: valosin containing protein; WT: wild-type.
Asunto(s)
Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Lisosomas , Fibras Musculares Esqueléticas/metabolismo , Proteína que Contiene Valosina/metabolismo , Animales , Autofagia/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Chaperón BiP del Retículo Endoplásmico , Células HeLa , Homeostasis/genética , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/genética , Lisosomas/patología , Lisosomas/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/ultraestructura , Proteína que Contiene Valosina/antagonistas & inhibidores , Proteína que Contiene Valosina/genéticaRESUMEN
Lysosomal membrane permeabilization or lysosomal rupture is recognized as a common and severe stress condition relevant for infection, cellular degeneration and cancer. However, the cellular response mechanisms that protect cells from the consequences of lysosomal damage and ensure lysosomal quality control and homeostasis have only recently been explored. Key elements of this response involve the specific sensing of the damage followed by extensive modification of the organelles with ubiquitin to mark them for clearance by selective macroautophagy, termed lysophagy. Efficient lysophagy is ensured by additional layers of regulation, including modulation by the ubiquitin-directed AAA-ATPase VCP/p97. Lysophagy shares many features with mitophagy, the macroautophagic removal of damaged mitochondria. This review aims to gather available data from different fields and to define the key steps necessary for sensing and subsequent clearance of damaged lysosomes. We conclude with a discussion of disease implications with a focus on neurodegeneration.
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Adenosina Trifosfatasas/metabolismo , Autofagia/fisiología , Membranas Intracelulares/fisiología , Lisosomas/fisiología , Enfermedades Neurodegenerativas/fisiopatología , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismoRESUMEN
Two missense mutations of the DYRK1B gene have recently been found to co-segregate with a rare autosomal-dominant form of metabolic syndrome. This gene encodes a member of the DYRK family of protein kinases, which depend on tyrosine autophosphorylation to acquire the catalytically active conformation. The mutations (H90P and R102C) affect a structural element named DYRK homology (DH) box and did not directly interfere with the conformation of the catalytic domain in a structural model of DYRK1B. Cellular assays showed that the mutations did not alter the specific activity of mature kinase molecules. However, a significant part of the mutant DYRK1B protein accumulated in detergent-insoluble cytoplasmic aggregates and was underphosphorylated on tyrosine. The mutant DYRK1B variants were more vulnerable to the HSP90 inhibitor ganetespib and showed enhanced binding to the co-chaperone CDC37 as compared to wild type DYRK1B. These results support the hypothesis that the mutations in the DH box interfere with the maturation of DYRK1B by tyrosine autophosphorylation and compromise the conformational stability of the catalytic domain, which renders the kinase susceptible to misfolding.
Asunto(s)
Síndrome Metabólico/genética , Mutación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Dominio Catalítico , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Células HeLa , Humanos , Fosforilación/efectos de los fármacos , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Triazoles/farmacología , Tirosina/metabolismo , Quinasas DyrKRESUMEN
Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.
Asunto(s)
Sistemas de Liberación de Medicamentos , Nanopartículas/administración & dosificación , Ficoeritrina/administración & dosificación , Proteolisis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Fosfatos de Calcio/administración & dosificación , Fosfatos de Calcio/química , Endocitosis , Endosomas/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Lisosomas/efectos de los fármacos , Macrólidos/administración & dosificación , Nanopartículas/química , Ficoeritrina/químicaRESUMEN
Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Autofagia , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endopeptidasas/metabolismo , Lisosomas/metabolismo , Proteínas/metabolismo , Tioléster Hidrolasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Animales , Proteínas Relacionadas con la Autofagia , Células Cultivadas , Humanos , Ratones , Proteína que Contiene ValosinaRESUMEN
Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). We now show that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin was sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolished LD targeting. Increased levels of spastin-M1 expression reduced the number but increased the size of LDs. Expression of a mutant unable to bind and sever MTs caused clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila led to bigger and less numerous LDs in the fat bodies and increased triacylglycerol levels. In contrast, Dspastin overexpression increased LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreased LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduced triacylglycerol levels in the larvae. Moreover, we found reduced amount of fat stores in intestinal cells of worms in which the spas-1 homologue was either depleted by RNA interference or deleted. Taken together, our data uncovers an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Drosophila/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Retículo Endoplásmico/metabolismo , Mucosa Intestinal/metabolismo , Músculo Esquelético/metabolismo , Neuronas/metabolismo , Transporte de Proteínas , Triglicéridos/metabolismoRESUMEN
Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.
Asunto(s)
Empalme Alternativo/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Especificidad de Órganos/fisiología , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/fisiología , Proteínas Tirosina Quinasas/genética , Especificidad por Sustrato/fisiología , Quinasas DyrKRESUMEN
DYRK1A is a dual-specificity protein kinase that autophosphorylates a conserved tyrosine residue in the activation loop but phosphorylates exogenous substrates only at serine or threonine residues. Tyrosine autophosphorylation of DYRKs is a one-off event that takes place during translation and induces the activation of the kinase. Here we characterize the beta-carboline alkaloid harmine as a potent and specific inhibitor of DYRK1A both in vitro and in cultured cells. Comparative in vitro assays of four kinases of the DYRK family showed that harmine inhibited substrate phosphorylation by DYRK1A more potently than it inhibited substrate phosphorylation by the closely related kinase DYRK1B [half maximal inhibitory concentrations (IC(50)) of 33 nm versus 166 nm, respectively] and by the more distant members of the family, DYRK2 and DYRK4 (1.9 microm and 80 microm, respectively). Much higher concentrations of harmine were required to suppress tyrosine autophosphorylation of the translational intermediate of DYRK1A in a bacterial in vitro translation system (IC(50) = 1.9 microm). Importantly, harmine inhibited the phosphorylation of a specific substrate by DYRK1A in cultured cells with a potency similar to that observed in vitro (IC(50) = 48 nm), without negative effects on the viability of the cells. Overexpression of the DYRK1A gene on chromosome 21 has been implicated in the altered neuronal development observed in Down syndrome. Here, we show that harmine interferes with neuritogenesis in cultured hippocampal neurons. In summary, our data show that harmine inhibits DYRK1A substrate phosphorylation more potently than it inhibits tyrosine autophosphorylation, and provide evidence for a role of DYRK1A in the regulation of neurite formation.