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The present study aimed to identify and verify new plasma protein markers to predict the female fecundability level. A nested case-control study was conducted involving couples who participated in the Chinese National Free Preconception Check-up Project. Women who successfully conceive within one year were defined as the high fecundability group, and those unable to conceive were defined as the low fecundability group. In the training cohort, potential protein biomarkers were identified using proteomics technology and were further tested in a validation cohort by the Western blotting assay, enzyme-linked immunosorbent assay, and biochemical tests. Meanwhile, receiver operating characteristic curve analysis were used to evaluate the predictive value. Cox proportional hazard regression analyses were conducted to calculate hazard ratios; restricted cubic spline analysis was used to assess the linear relationship between the the protein level and hazard ratios for fecundability. Pyruvate, a key product of glycolysis, was significantly increased in the high fecundability group (P < 0.01) compared to the low fecundability group, and its area under the curve value was 0.68 (P < 0.05). There was a linear positive dose-response association between the pyruvate level and fecundability possibility (hazard ratios = 1.66, 95% CI: 1.07-2.59, p for trend = 0.025, nonlinearity, p-value = 0.2927).
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This study aims to investigate the clinical characteristics of infections following induction chemotherapy for acute myeloid leukemia (AML) in children and identify risk factors associated with severe infections. Newly diagnosed children with AML treated at the Hematology Oncology Center of Beijing Children's Hospital affiliated to Capital Medical University (referred to as the "Beijing ward") and Baoding Hospital of Beijing Children's Hospital (referred to as the "Baoding ward") between November 2019 and August 2022 were enrolled. The diagnosis and treatment of the patients were carried out in accordance with the CCLG-AML 2019 protocol. Their essential information and infection-related indicators were collected. The various indicators between the two centers were compared. The incidence of infection in children with AML was 100%, with a severe infection rate of 15.3% and an infection-related mortality rate of 2.4%. Respiratory infections accounted for 39.7% of all infections events, and unspecified site infection for 32.2%. Bacterial infections were predominant at 51.2%. The bed unit area (BUA) varied significantly with 4.1 m2 in the Beijing ward and 10 m2 in the Baoding ward. Significant differences were observed in gastrointestinal infections (P < 0.001) and severe infections (P = 0.014) between the two wards. Several factors were identified as risk factors for severe infections, including BUA difference (OR = 4.353, 95% CI: 1.078-17.578), time of entering neutropenia phase after chemotherapy (OR = 6.369, 95% CI: 1.713-23.675) and bloodstream infection (OR = 7.466, 95% CI: 1.889-29.507). Respiratory tract infections and infections of unspecified site are most common during induction phase for pediatric AML. Bacteria, especially G- bacteria, are the leading pathogens. Risk factors for severe infections include a small BUA, entering neutropenia phase ≤ 5.5 days after chemotherapy, and bloodstream infection. Recognizing these risk factors can aid in the early identification and intervention of severe infections.
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Finding a low-cost and highly efficient method for identifying subway tunnel damage can greatly reduce catastrophic accidents. At present, tunnel health monitoring is mainly based on the observation of apparent diseases and vibration monitoring, which is combined with a manual inspection to perceive the tunnel health status. However, these methods have disadvantages such as high cost, short working time, and low identification efficiency. Thus, in this study, a tunnel damage identification algorithm based on the vibration response of in-service train and WPE-CVAE is proposed, which can automatically identify tunnel damage and give the damage location. The method is an unsupervised novelty detection that requires only sufficient normal data on healthy structure for training. This study introduces the theory and implementation process of this method in detail. Through laboratory model tests, the damage of the void behind the tunnel wall is designed to verify the performance of the algorithm. In the test case, the proposed method achieves the damage identification performance with a 96.25% recall rate, 86.75% hit rate, and 91.5% accuracy. Furthermore, compared with the other unsupervised methods, the method performance and noise immunity are better than others, so it has a certain practical value.
Assuntos
Algoritmos , Análise de OndaletasRESUMO
As an important part of urban rail transit, subway tunnels play an important role in alleviating traffic pressure in mega-cities. Identifying and locating damage to the tunnel structure as early as possible has important practical significance for maintaining the long-term safe operation of subway tunnels. Summarizing the current status and shortcomings of the structural health monitoring of subway tunnels, a very economical and effective monitoring program is proposed, which is to use the train vibration response to identify and locate the damage of the tunnel structure. Firstly, the control equation of vehicle-tunnel coupling vibration is established and its analytical solution is given as the theoretical basis of this paper. Then, a damage index based on the cumulative sum of wavelet packet energy change rate (TDISC) is proposed, and its process algorithm is given. Through the joint simulation of VI-Rail and ANSYS, a refined 3D train-tunnel coupled vibration model is established. In this model, different combined conditions of single damage and double damage verify the validity of the damage index. The effectiveness of this damage index was further verified through model tests, and the influence of vehicle speed and load on the algorithm was discussed. Numerical simulation and experimental results show that the TDISC can effectively locate the damage of the tunnel structure and has good robustness.
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Ferrovias , Cidades , VibraçãoRESUMO
Protein aggregates containing ubiquitin (Ub) are commonly observed in neurodegenerative disorders, implicating the involvement of the ubiquitin proteasome system (UPS) in their pathogenesis. Here, we aimed to generate a mouse model for monitoring UPS function using a green fluorescent protein (GFP)-based substrate that carries a "noncleavable" N-terminal ubiquitin moiety (Ub(G76V)). We engineered transgenic mice expressing a fusion protein, consisting of the following: (1) Ub(G76V), GFP, and a synaptic vesicle protein synaptobrevin-2 (Ub(G76V)-GFP-Syb2); (2) GFP-Syb2; or (3) Ub(G76V)-GFP-Syntaxin1, all under the control of a neuron-specific Thy-1 promoter. As expected, Ub(G76V)-GFP-Syb2, GFP-Syb2, and Ub(G76V)-GFP-Sytaxin1 were highly expressed in neurons, such as motoneurons and motor nerve terminals of the neuromuscular junction (NMJ). Surprisingly, Ub(G76V)-GFP-Syb2 mice developed progressive adult-onset degeneration of motor nerve terminals, whereas GFP-Syb2 and Ub(G76V)-GFP-Syntaxin1 mice were normal. The degeneration of nerve terminals in Ub(G76V)-GFP-Syb2 mice was preceded by a progressive impairment of synaptic transmission at the NMJs. Biochemical analyses demonstrated that Ub(G76V)-GFP-Syb2 interacted with SNAP-25 and Syntaxin1, the SNARE partners of synaptobrevin. Ultrastructural analyses revealed a marked reduction in synaptic vesicle density, accompanying an accumulation of tubulovesicular structures at presynaptic nerve terminals. These morphological defects were largely restricted to motor nerve terminals, as the ultrastructure of motoneuron somata appeared to be normal at the stages when synaptic nerve terminals degenerated. Furthermore, synaptic vesicle endocytosis and membrane trafficking were impaired in Ub(G76V)-GFP-Syb2 mice. These findings indicate that Ub(G76V)-GFP-Syb2 may compete with endogenous synaptobrevin, acting as a gain-of-function mutation that impedes SNARE function, resulting in the depletion of synaptic vesicles and degeneration of the nerve terminals. SIGNIFICANCE STATEMENT: Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (Ub(G76V)-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration.
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Doença dos Neurônios Motores/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Ubiquitina/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Doença dos Neurônios Motores/patologia , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ubiquitina/genéticaRESUMO
Friedreich ataxia is an early-onset multisystemic disease linked to a variety of molecular defects in the nuclear gene FRDA. This gene normally encodes the iron-binding protein frataxin (FXN), which is critical for mitochondrial iron metabolism, global cellular iron homeostasis, and antioxidant protection. In most Friedreich ataxia patients, a large GAA-repeat expansion is present within the first intron of both FRDA alleles, that results in transcriptional silencing ultimately leading to insufficient levels of FXN protein in the mitochondrial matrix and probably other cellular compartments. The lack of FXN in turn impairs incorporation of iron into iron-sulfur cluster and heme cofactors, causing widespread enzymatic deficits and oxidative damage catalyzed by excess labile iron. In a minority of patients, a typical GAA expansion is present in only one FRDA allele, whereas a missense mutation is found in the other allele. Although it is known that the disease course for these patients can be as severe as for patients with two expanded FRDA alleles, the underlying pathophysiological mechanisms are not understood. Human cells normally contain two major mitochondrial isoforms of FXN (FXN(42-210) and FXN(81-210)) that have different biochemical properties and functional roles. Using cell-free systems and different cellular models, we show that two of the most clinically severe FXN point mutations, I154F and W155R, have unique direct and indirect effects on the stability, biogenesis, or catalytic activity of FXN(42-210) and FXN(81-210) under physiological conditions. Our data indicate that frataxin point mutations have complex biochemical effects that synergistically contribute to the pathophysiology of Friedreich ataxia.
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Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Alelos , Substituição de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Sistema Livre de Células , Chlorocebus aethiops , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Humanos , Proteínas de Ligação ao Ferro/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Expansão das Repetições de Trinucleotídeos , FrataxinaRESUMO
This paper reports a new solid self-emulsifying drug delivery system--self-emulsifying microsphere prepared by membrane emulsification technology with the hydrophobic berberine hydrochloride as a model drug. Solubility test and pseudo-ternary phase diagram were employed to select the optimal prescription of liquid self-emulsifying drug delivery system. The self-emulsifying microsphere was prepared by membrane emulsification technology with the solid carrier in a gel formed by sodium alginate and calcium chloride. The results showed that the optimal prescription of liquid self-emulsifying drug delivery system was Capmul MCM/Cremophor RH40/Labrasol/1-2propanediol = 20 : 32 : 32 : 16. The solid self-emulsifying microsphere had average diameter of 10.92 microm, encapsulation efficiency of 32.57% and the droplet size of reconstituted micromulsion of 156.5 nm. Berberine hydrochloride was dispersed in microsphere in non-crystalline form. In vitro release of the self-emulsifying microsphere showed pH response characteristics. These results indicated that the self-emulsifying microsphere prepared by membrane emulsification technology might become a new dosage form for poorly water soluble drugs.
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Berberina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Tecnologia Farmacêutica/métodos , Berberina/química , Emulsões , Microesferas , Tamanho da Partícula , SolubilidadeRESUMO
Fe-S clusters (ISCs) are versatile cofactors utilized by many mitochondrial, cytoplasmic, and nuclear enzymes. Whereas mitochondria can independently initiate and complete ISC synthesis, other cellular compartments are believed to assemble ISCs from putative precursors exported from the mitochondria via an ATP binding cassette (ABC) transporter conserved from yeast (Atm1p) to humans (ABCB7). However, the regulatory interactions between mitochondrial and extramitochondrial ISC synthesis are largely unknown. In yeast, we found that mitochondrial ISC synthesis is regulated by the leucine biosynthetic pathway, which among other proteins involves an abundant cytoplasmic [4Fe-4S] enzyme, Leu1p. Enzymatic blocks in the pathway (i.e. leu1Δ or leu2Δ gene deletion mutations) induced post-transcriptional up-regulation of core components of mitochondrial ISC biosynthesis (i.e. the sulfur donor Nfs1p, the iron donor Yfh1p, and the ISC scaffold Isu1p). In leu2Δ cells, transcriptional mechanisms also led to dramatic up-regulation of Leu1p with concomitant down-regulation of mitochondrial aconitase (Aco1p), a [4Fe-4S] enzyme in the tricarboxylic acid cycle. Accordingly, the leu2Δ deletion mutation exacerbated Aco1p inactivation in cells with mutations in Yfh1p. These data indicate that defects in leucine biosynthesis promote the biogenesis of enzymatically active Leu1p at the expense of Aco1p activity. Surprisingly, this effect is independent of Atm1p; previous reports linking the loss of Leu1p activity to Atm1p depletion were confounded by the fact that LEU2 was used as a selectable marker to create Atm1p-depleted cells, whereas a leu2Δ allele was present in Atm1p-repleted controls. Thus, still largely unknown transcriptional and post-transcriptional mechanisms control ISC distribution between mitochondria and other cellular compartments.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Citoplasma/metabolismo , Proteína 1 Reguladora do Ferro/biossíntese , Leucina/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Respiração Celular , DNA Fúngico/metabolismo , Regulação para Baixo , Proteína 1 Reguladora do Ferro/metabolismo , Mitocôndrias/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: For cultivation and high yield of oilseed rape (Brassica napus L.) in China, traditional seedling transplanting is replaced by seed-sowing but, better nitrogen management is crucial and not established yet. This study aimed to adapt N management to the seed-sowing method for the winter oilseed rape and to minimize the N fertilizer-derived pollution potential in the upper reaches of Yangtze River Basin. MATERIALS AND METHODS: Three field experiments were conducted to check effect of different doses of N fertilizers, split doses of N and different types of N fertilizers for seed-sowing winter oilseed rape with high plant density in upper reaches of Yangtze River Basin in Sichuan province of China. RESULTS: In first experiment, among four doses (0, 90, 180 and 270 kg N ha-1) on average 3.54 t ha-1 was in 180 kg N ha-1 and 3.61 t ha-1 in 270 kg N ha-1 while cultivars dy6 and cn3 produced 3.23 and 3.29 t ha-1 which is significantly higher than zs11. There was no significant difference in N-use efficiency among three cultivars tested and second experiment showed no significant difference in seed yield with split N application. The third experiment compared the effects of different fertilizer types (urea, coated urea, 1:1 mixture of urea and coated urea and compound nitrogen fertilizer) on seed yield and get no significant difference in seed yield. CONCLUSION: This experiment proved that seed sowing method with higher nitrogen had high yield in the upper reaches of Yangtze River Basin in China, but higher N application may cause environment pollution. So, seed sowing method with nitrogen 180 kg N ha-1 was proved to be more effective.
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Agricultura/métodos , Brassica napus/crescimento & desenvolvimento , Fertilizantes , Nitrogênio/química , Sementes/crescimento & desenvolvimento , China , Poluentes Ambientais/química , Brotos de Planta/crescimento & desenvolvimento , Rios , Estações do Ano , Solo , Poluentes do SoloRESUMO
In the plant pathogen Pseudomonas syringae, production of the exopolysaccharide levan is mediated by extracellular levansucrase (Lsc), which is encoded by two functional genes, lscB and lscC. Comparison of extracellular protein profiles of P. syringae pv. glycinea PG4180 grown at 18 and 28 degrees C and Western blots revealed that Lsc was predominantly found in the supernatant at 18 degrees C, a temperature fostering virulence of this pathogen. Northern blot analysis indicated that transcription of lscB and lscC was temperature-dependent. Quantification of Lsc in supernatants and cellular protein samples of mutants defective in either lscB or lscC confirmed that LscB secretion at low temperature was due to a combination of thermo-regulated transcription and secretion. In contrast, LscC accumulated in the periplasmic space. LscB and LscC differ in only five amino acid residues, one of which is a cysteine residue. Temperature shift experiments suggested that de novo synthesized protein(s) at 18 degrees C might be responsible for differential LscB secretion and that the presumed secretory machinery was stable when cells were shifted to 28 degrees C. Our results imply that Lsc export and secretion may occur by yet-to-be identified novel mechanism(s).
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Regulação Bacteriana da Expressão Gênica/fisiologia , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Pseudomonas syringae/enzimologia , Northern Blotting , Western Blotting , Temperatura Alta , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidade , Glycine max/microbiologiaRESUMO
Mitochondrial biosynthesis of iron-sulfur clusters (ISCs) is a vital process involving the delivery of elemental iron and sulfur to a scaffold protein via molecular interactions that are still poorly defined. Analysis of highly conserved components of the yeast ISC assembly machinery shows that the iron-chaperone, Yfh1, and the sulfur-donor complex, Nfs1-Isd11, directly bind to each other. This interaction is mediated by direct Yfh1-Isd11 contacts. Moreover, both Yfh1 and Nfs1-Isd11 can directly bind to the scaffold, Isu1. Binding of Yfh1 to Nfs1-Isd11 or Isu1 requires oligomerization of Yfh1 and can occur in an iron-independent manner. However, more stable contacts are formed when Yfh1 oligomerization is normally coupled with the binding and oxidation of Fe2+. Our observations challenge the view that iron delivery for ISC synthesis is mediated by Fe2+-loaded monomeric Yfh1. Rather, we find that the iron oxidation-driven oligomerization of Yfh1 promotes the assembly of stable multicomponent complexes in which the iron donor and the sulfur donor simultaneously interact with each other as well as with the scaffold. Moreover, the ability to store ferric iron enables oligomeric Yfh1 to adjust iron release depending on the presence of Isu1 and the availability of elemental sulfur and reducing equivalents. In contrast, the use of anaerobic conditions that prevent Yfh1 oligomerization results in inhibition of ISC assembly on Isu1. These findings suggest that iron-dependent oligomerization is a mechanism by which the iron donor promotes assembly of the core machinery for mitochondrial ISC synthesis.
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Proteínas de Ligação ao Ferro/química , Proteínas Ferro-Enxofre/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiologia , Sulfurtransferases/química , Cromatografia em Gel , DNA/química , Relação Dose-Resposta a Droga , Regulação Fúngica da Expressão Gênica , Ferro/química , Modelos Biológicos , Ligação Proteica , Fatores de Tempo , FrataxinaRESUMO
Formation of the vertebrate neuromuscular junction (NMJ) takes place in a stereotypic pattern in which nerves terminate at select sarcolemmal sites often localized to the central region of the muscle fibers. Several lines of evidence indicate that the muscle fibers may initiate postsynaptic differentiation independent of the ingrowing nerves. For example, nascent acetylcholine receptors (AChRs) are pre-patterned at select regions of the muscle during the initial stage of neuromuscular synaptogenesis. It is not clear how these pre-patterned AChR clusters are assembled, and to what extent they contribute to pre- and post-synaptic differentiation during development. Here, we show that genetic deletion of the AChR gamma-subunit gene in mice leads to an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis. The absence of pre-patterned AChR clusters was associated with excessive nerve branching, increased motoneuron survival, as well as aberrant distribution of acetylcholinesterase (AChE) and rapsyn. However, clustering of muscle specific kinase (MuSK) proceeded normally in the gamma-null muscles. AChR clusters emerged at later stages owing to the expression of the AChR epsilon-subunit, but these delayed AChR clusters were broadly distributed and appeared at lower level compared with the wild-type muscles. Interestingly, despite the abnormal pattern, synaptic vesicle proteins were progressively accumulated at individual nerve terminals, and neuromuscular synapses were ultimately established in gamma-null muscles. These results demonstrate that the gamma-subunit is required for the formation of pre-patterned AChR clusters, which in turn play an essential role in determining the subsequent pattern of neuromuscular synaptogenesis.
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Junção Neuromuscular/metabolismo , Organogênese/fisiologia , Receptores Colinérgicos/fisiologia , Acetilcolinesterase/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Eletrofisiologia , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Proteínas Musculares/metabolismo , Músculos/embriologia , Músculos/metabolismo , Junção Neuromuscular/embriologia , Junção Neuromuscular/genética , Organogênese/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.
Assuntos
Biofilmes/crescimento & desenvolvimento , Frutanos/biossíntese , Polissacarídeos Bacterianos/fisiologia , Pseudomonas syringae/fisiologia , Alginatos/análise , Fluorescência , Frutanos/análise , Frutanos/genética , Deleção de Genes , Ácido Glucurônico/análise , Ácido Glucurônico/biossíntese , Ácido Glucurônico/genética , Ácido Glucurônico/fisiologia , Hexosiltransferases/análise , Ácidos Hexurônicos/análise , Lectinas/metabolismo , Microscopia Confocal , Polissacarídeos Bacterianos/análise , Polissacarídeos Bacterianos/biossíntese , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Coloração e RotulagemRESUMO
Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Encéfalo/metabolismo , Linhagem Celular , Dimerização , Endopeptidases/metabolismo , Genes Reporter , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Peptídeo Hidrolases , Presenilina-1 , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ratos , Receptores Notch , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein and signaling receptor Notch. PS holoprotein undergoes endoproteolysis by an unknown enzymatic activity to generate NH(2)- and COOH-terminal fragments, a process that is required for the formation of the active and stable PS/-gamma-secretase complex. Biochemical and genetic studies have recently identified nicastrin, APH-1, and PEN-2 as essential cofactors that physically interact with PS1 and are necessary for the gamma-secretase activity. However, their precise function in regulating the PS complex and gamma-secretase activity remains unknown. Here, we demonstrate that endogenous PEN-2 preferentially interacts with PS1 holoprotein. Down-regulation of PEN-2 expression by small interfering RNA (siRNA) abolishes the endoproteolysis of PS1, whereas overexpression of PEN-2 promotes the production of PS1 fragments, indicating a critical role for PEN-2 in PS1 endoproteolysis. Interestingly, accumulation of full-length PS1 resulting from down-regulation of PEN-2 is alleviated by additional siRNA down-regulation of APH-1. Furthermore, overexpression of APH-1 facilitates PEN-2-mediated PS1 proteolysis, resulting in a significant increase in PS1 fragments. Our data reveal a direct role of PEN-2 in proteolytic cleavage of PS1 and a regulatory function of APH-1, in coordination with PEN-2, in the biogenesis of the PS1 complex.