RESUMEN
Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. However, structural and functional properties of some CBMs remain unknown, and it is not clear why some LPMOs, like CjLPMO10A from the soil bacterium Cellvibrio japonicus, have multiple CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on their functional variation and determined the solution structures of both by NMR, which constitutes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the micromolar range, CjCBM73 has higher affinity for chitin than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding of CjCBM73 to this chitooligosaccharide was detected. These functional differences correlate with distinctly different arrangements of three conserved aromatic amino acids involved in substrate binding. In CjCBM5, these residues show a linear arrangement that seems compatible with the experimentally observed affinity for single chitin chains. On the other hand, the arrangement of these residues in CjCBM73 suggests a wider binding surface that may interact with several chitin chains. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.
Asunto(s)
Proteínas Bacterianas/química , Cellvibrio/enzimología , Quitosano/química , Oxigenasas de Función Mixta/química , Oligosacáridos/química , Dominios ProteicosRESUMEN
Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and ß-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants-JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18-indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.
Asunto(s)
Actinobacteria/enzimología , Quitinasas/metabolismo , Actinobacteria/metabolismo , Proteínas Bacterianas/metabolismo , Catálisis , Celulosa/metabolismo , Quitina/metabolismo , Glicósido Hidrolasas/metabolismo , Glicósidos/metabolismo , Hidrólisis , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , Estrés Oxidativo/fisiología , Polisacáridos/metabolismo , Especificidad por SustratoRESUMEN
Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.
Asunto(s)
Complejos de Coordinación , Esterasas , Estereoisomerismo , Oxidación-Reducción , CatálisisRESUMEN
Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane trafficking, but disease mechanisms remain unclear. We show that OCRL visits late-stage, endocytic clathrin-coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes. The interaction with APPL1, which is mediated by the ASH-RhoGAP-like domains of OCRL and is abolished by disease mutations, provides a link to protein networks implicated in the reabsorptive function of the kidney and in the trafficking and signaling of growth factor receptors in the brain. Crystallographic studies reveal a role of the ASH-RhoGAP-like domains in positioning the phosphatase domain at the membrane interface and a clathrin box protruding from the RhoGAP-like domain. Our results support a role of OCRL in the early endocytic pathway, consistent with the predominant localization of its preferred substrates, PI(4,5)P(2) and PI(3,4,5)P(3), at the cell surface.
Asunto(s)
Endocitosis/fisiología , Endosomas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Chlorocebus aethiops , Vesículas Cubiertas por Clatrina/metabolismo , Cristalografía por Rayos X , Endosomas/enzimología , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Riñón/citología , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kbeta, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinasebeta (PI3Kbeta), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.
Asunto(s)
Endocitosis , Fosfatidilinositoles/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/metabolismo , Catálisis , Compartimento Celular , Cromatografía de Afinidad , Regulación hacia Abajo/genética , Activación Enzimática , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Unión Proteica , Transporte de Proteínas , Suero , Transferrina/metabolismo , Proteínas de Unión al GTP rab5/aislamiento & purificaciónRESUMEN
A key step in glutamatergic synapse maturation is the replacement of developmentally expressed N-methyl-D-aspartate receptors (NMDARs) with mature forms that differ in subunit composition, electrophysiological properties and propensity to elicit synaptic plasticity. However, the mechanisms underlying the removal and replacement of synaptic NMDARs are poorly understood. Here we demonstrate that NMDARs containing the developmentally regulated NR3A subunit undergo rapid endocytosis from the dendritic plasma membrane in cultured rat hippocampal neurons. This endocytic removal is regulated by PACSIN1/syndapin1, which directly and selectively binds the carboxy-terminal domain of NR3A through its NPF motifs and assembles a complex of proteins including dynamin and clathrin. Endocytosis of NR3A by PACSIN1 is activity dependent, and disruption of PACSIN1 function causes NR3A accumulation at synaptic sites. Our results reveal a new activity-dependent mechanism involved in the regulation of NMDAR expression at synapses during development, and identify a brain-specific endocytic adaptor that confers spatiotemporal and subunit specificity to NMDAR endocytosis.
Asunto(s)
Endocitosis/fisiología , Glicoproteínas de Membrana/fisiología , Neuronas/citología , Proteoglicanos/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Western Blotting/métodos , Células Cultivadas , Clonación Molecular/métodos , Electrofisiología/métodos , Embrión de Mamíferos , Endocitosis/efectos de los fármacos , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Humanos , Hibridación in Situ/métodos , Glicoproteínas de Membrana/genética , Microscopía Inmunoelectrónica/métodos , Mutación/fisiología , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Proteoglicanos/genética , Ratas , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructura , Sindecanos , Transfección/métodos , Técnicas del Sistema de Dos HíbridosRESUMEN
We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Células 3T3 , Aciltransferasas/química , Aciltransferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Western Blotting , Encéfalo/enzimología , ADN Complementario , Ácidos Grasos/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismoRESUMEN
Huntington's disease (HD) is caused by a pathological expansion of a CAG repeat in the first exon of the gene coding for huntingtin, resulting in an abnormally long polyglutamine stretch. Despite its widespread expression, mutant huntingtin leads to selective neuronal loss in the striatum and cortex. Here we report that the neurospecific phosphoprotein PACSIN 1, which has been implicated as playing a central role in synaptic vesicle recycling, interacts with huntingtin via its C-terminal SH3 domain. Moreover, two other isoforms, PACSIN 2 and 3, which show a wider tissue distribution including the brain, do not interact with huntingtin despite a highly conserved SH3 domain. Furthermore, this interaction is repeat-length-dependent and is enhanced with mutant huntingtin, possibly causing the sequestration of PACSIN 1. Normally, PACSIN 1 is located along neurites and within synaptic boutons, but in HD patient neurons, there is a progressive loss of PACSIN 1 immunostaining in synaptic varicosities, beginning in presymptomatic and early-stage HD. Further, PACSIN 1 immunostaining of HD patient tissue reveals a more cytoplasmic distribution of the protein, with particular concentration in the perinuclear region coincident with mutant huntingtin. Thus, the specific interaction of huntingtin with the neuronal PACSIN isoform, PACSIN 1, and its altered intracellular distribution in pathological tissue, together with the observed differences in the binding behavior, suggest a role for PACSIN 1 during early stages of the selective neuropathology of HD.