RESUMEN
Tamoxifen, the gold standard drug for endocrine therapy for breast cancer, modulates the phosphorylation status of the TAU protein in Alzheimer's disease by inhibiting CDK5 kinase activity. Its binding to p25 prevents CDK5/p25 complexation and hence a decrease of CDK5 activity. In breast tumors, this complex is involved in the proliferation and survival of cancer cells, as well as in the disease's prognosis. Still, the molecular stability of the CDK5/p25 complex following tamoxifen exposure in this cancer type has not yet been clearly deciphered. Here, we report the functional characterization of CDK5 and its p25 regulatory subunit in the absence and presence of tamoxifen. In addition, two novel inhibitors of the kinase activity of the CDK5/p25 complex are identified, both of which would reduce the risk of recurrence of estrogen receptor-positive (ER+) breast cancers and prevent drawbacks induced by tamoxifen exposure. Accordingly, 6His-CDK5 and 6His-p25 have been expressed and purified. Fluorescence anisotropy measurements have been used to assess that the two proteins do form an active complex, and thermodynamic parameters of their interaction were measured. It was also confirmed that tamoxifen directly binds to p25 and inhibits CDK5 kinase activity. Similar observations were obtained using 4-hydroxytamoxifen, an active metabolized form of tamoxifen. Two novel compounds have been identified here that harbor a benzofuran moiety and were shown to target directly p25, and their bindings resulted in decreased CDK5 kinase activity. This encouraging alternative opens the way to the ensuing chemical optimization of this scaffold. It also promises a more specific therapeutic approach that may both tackle the pathological signaling in breast cancer and provide a potential new drug for Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer , Neoplasias de la Mama , Humanos , Femenino , Fosforilación , Enfermedad de Alzheimer/metabolismo , Proteínas tau/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Transducción de Señal , TamoxifenoRESUMEN
Lactoferrin is a multifunctional glycoprotein found in the milk of most mammals. In addition to its well-known role of binding iron, lactoferrin carries many important biological functions, including the promotion of cell proliferation and differentiation, and as an anti-bacterial, anti-viral, and anti-parasitic protein. These functions differ among lactoferrin homologs in mammals. Although considerable attention has been given to the many functions of lactoferrin, its primary nutritional contribution is presumed to be related to its iron-binding characteristics, whereas the role of glycosylation has been neglected. Given the critical role of glycan binding in many biological processes, the glycan moieties in lactoferrin are likely to contribute significantly to the biological roles of lactoferrin. Despite the high amino acid sequence homology in different lactoferrins (up to 99%), each exhibits a unique glycosylation pattern that may be responsible for heterogeneity of the biological properties of lactoferrins. An important task for the production of biotherapeutics and medical foods containing bioactive glycoproteins is the assessment of the contributions of individual glycans to the observed bioactivities. This review examines how the study of lactoferrin glycosylation patterns can increase our understanding of lactoferrin functionality.
Asunto(s)
Lactoferrina/metabolismo , Animales , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Humanos , Lactoferrina/química , Espectrometría de Masas , Proteínas de la Leche/química , Proteínas de la Leche/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad ProteicaRESUMEN
In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.
Asunto(s)
Retículo Endoplásmico Rugoso/metabolismo , Retículo Endoplásmico Liso/metabolismo , Lactancia/metabolismo , Glándulas Mamarias Animales/metabolismo , Animales , Biomarcadores/metabolismo , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Retículo Endoplásmico Rugoso/ultraestructura , Retículo Endoplásmico Liso/ultraestructura , Epitelio/metabolismo , Epitelio/ultraestructura , Femenino , Cabras , Glándulas Mamarias Animales/ultraestructura , Microscopía Electrónica de Transmisión , Microsomas/metabolismo , Microsomas/ultraestructura , Ratas , Especificidad de la Especie , Factores de TiempoRESUMEN
UNLABELLED: Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-ß-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides. IMPORTANCE: It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip-quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate.
Asunto(s)
Bifidobacterium/enzimología , Bifidobacterium/metabolismo , Glicoproteínas/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Leche/metabolismo , Oligosacáridos/metabolismo , Animales , Bifidobacterium/crecimiento & desarrollo , Humanos , LactanteRESUMEN
Numerous milk components, such as lactoferrin, are recognized as health-promoting compounds. A growing body of evidence suggests that glycans could mediate lactoferrin's bioactivity. Goat milk lactoferrin is a candidate for infant formula supplementation because of its high homology with its human counterpart. The aim of this study was to characterize the glycosylation pattern of goat milk lactoferrin. After the protein was isolated from milk by affinity chromatography, N-glycans were enzymatically released and a complete characterization of glycan composition was carried out by advanced MS. The glycosylation of goat milk lactoferrin was compared with that of human and bovine milk glycoproteins. Nano-LC-Chip-Q-TOF MS data identified 65 structures, including high mannose, hybrid, and complex N-glycans. Among the N-glycan compositions, 37% were sialylated and 34% were fucosylated. The results demonstrated the existence of similar glycans in human and goat milk but also identified novel glycans in goat milk that were not present in human milk. These data suggest that goat milk could be a source of bioactive compounds, including lactoferrin that could be used as functional ingredients for food products beneficial to human nutrition.
Asunto(s)
Glicómica/métodos , Lactoferrina/análisis , Leche/química , Polisacáridos/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Cabras , Humanos , Datos de Secuencia Molecular , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. RESULTS: In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both alphaS1- and beta-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature beta-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature alphaS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of alphaS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of alphaS1-casein with membranes. CONCLUSIONS: These experiments reveal for the first time the existence of a membrane-associated form of alphaS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that alphaS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway.
Asunto(s)
Caseínas/metabolismo , Retículo Endoplásmico/metabolismo , Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Células Cultivadas , Células Epiteliales/citología , Femenino , Cabras , Humanos , Glándulas Mamarias Animales/citología , Multimerización de Proteína , Transporte de Proteínas , RatasRESUMEN
Donor milk is the best option when mother's own milk is unavailable. Heat treatments are applied to ensure donor milk safety. The effects of heat treatments on milk gangliosides-bioactive compounds with beneficial antibacterial, anti-inflammatory, and prebiotic roles-have not been studied. The most abundant gangliosides in non-homogenized human milk were characterized and quantified by liquid chromatography-mass spectrometry (LC-MS)/MS before and after pasteurization treatments mimicking industrial conditions (63 °C/30 min, 72 °C/15 s, 127 °C/5 s, and 140 °C/6 s). Ganglioside stability over a 3-month period was assessed following the storage at 4 and 23 °C. Independent of the heat treatment applied, gangliosides were stable after 3 months of storage at 4 or 23 °C, with only minor variations in individual ganglioside structures. These findings will help to define the ideal processing and storage conditions for donor milk to maximize the preservation of the structure of bioactive compounds to enhance the health of fragile newborns. Moreover, these results highlight the need for, and provide a basis for, a standardized language enabling biological and food companies, regulatory agencies, and other food stakeholders to both annotate and compute the ways in which production, processing, and storage conditions alter or maintain the nutritive, bioactive, and organoleptic properties of ingredients and foods, as well as the qualitative effects these foods and ingredients may have on conferring phenotype in the consuming organism.
RESUMEN
Endo-ß-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N'-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid, and complex N-glycans. These conjugated N-glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45-8.45), temperature (27.5-77.5°C), reaction time (15-475 min), and enzyme/protein ratio (1:3,000-1:333) were evaluated on the release of N-glycans from bovine colostrum whey by EndoBI-1. A central composite design was used, including a two-level factorial design (2(4)) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N-glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan-free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI-1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies.
Asunto(s)
Bifidobacterium/enzimología , Productos Lácteos/análisis , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Polisacáridos/química , Animales , Bovinos , Calostro/química , Glicoproteínas/química , Calor , Concentración de Iones de HidrógenoRESUMEN
Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, we have used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Tamoxifeno/metabolismo , Proteínas tau/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Sitios de Unión , Proteínas de Ciclo Celular/química , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Simulación del Acoplamiento Molecular , Proteínas del Tejido Nervioso/química , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas , Ratas Sprague-Dawley , Tamoxifeno/químicaRESUMEN
Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1)-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1)-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1)-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1)-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1)-casein. These experiments reveal that the insolubility of α(s1)-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1)-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.
Asunto(s)
Caseínas/química , Colesterol/química , Mamíferos/metabolismo , Microdominios de Membrana/química , Animales , Transporte Biológico , Caseínas/metabolismo , Detergentes/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Evolución Molecular , Femenino , Lactancia , Microdominios de Membrana/efectos de los fármacos , Micelas , Ratas Wistar , Especificidad de la EspecieRESUMEN
Beta-casein (beta-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However, the elements determining its micellization behavior in solution and interfacial behavior at the air-water interface are not well known. In order to study how the forced dimerisation influences functional properties of beta-CN, recombinant wild-type beta-CN was produced and distal cysteinylated forms of recombinant beta-CN were engineered. We show that 1) cysteinylated beta-CN formed mainly dimers bridged by disulfide bonds; 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible; 3) covalent disulfide linkage forms at the air-water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of beta-CN.