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1.
J Biol Chem ; : 107510, 2024 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-38944120

RESUMEN

The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the predominant ß-secretase, cleaving the amyloid precursor protein (APP) via the amyloidogenic pathway. In addition, BACE1 as an amyloid degrading enzyme (ADE), cleaves Aß to produce the C-terminally truncated non-toxic Aß fragment Aß34 which is an indicator of amyloid clearance. Here, we analyzed effects of BACE1 inhibitors on its opposing enzymatic functions, i.e., amyloidogenic (Aß producing) and amyloidolytic (Aß degrading) activities, using cell culture models with varying BACE1/APP ratios. Under high level BACE1 expression, low-dose inhibition unexpectedly yielded a two-fold increase in Aß42 and Aß40 levels. The concomitant decrease in Aß34 and secreted APPß levels suggested that the elevated Aß42 and Aß40 levels were due to the attenuated Aß degrading activity of BACE1. Notably, the amyloidolytic activity of BACE1 was impeded at lower BACE1 inhibitor concentrations compared to its amyloidogenic activity, thereby suggesting that the Aß degrading activity of BACE1 was more sensitive to inhibition than its Aß producing activity. Under endogenous BACE1 and APP levels, "low-dose" BACE1 inhibition affected both the Aß producing and degrading activities of BACE1, i.e., significantly increased Aß42/Aß40 ratio and decreased Aß34 levels, respectively. Further, we incubated recombinant BACE1 with synthetic Aß peptides and found that BACE1 has higher affinity for Aß substrates over APP. In summary, our results suggest that stimulating BACE1's ADE activity and halting Aß production without decreasing Aß clearance could still be a promising therapeutic approach with new, yet to be developed, BACE1 modulators.

2.
Mol Cell ; 84(12): 2368-2381.e6, 2024 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-38834067

RESUMEN

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.


Asunto(s)
Adenosina Difosfato , Adenosina Trifosfato , Microscopía por Crioelectrón , Elementos Transponibles de ADN , Transposasas , Elementos Transponibles de ADN/genética , Adenosina Trifosfato/metabolismo , Transposasas/metabolismo , Transposasas/genética , Transposasas/química , Adenosina Difosfato/metabolismo , Unión Proteica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Modelos Moleculares , Multimerización de Proteína , Sitios de Unión
3.
bioRxiv ; 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38798656

RESUMEN

The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo . First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX's effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX's dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae , but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

4.
J Biol Chem ; 300(1): 105465, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37979915

RESUMEN

Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.


Asunto(s)
Calreticulina , Plasminógeno , Animales , Bovinos , Humanos , Calreticulina/genética , Calreticulina/aislamiento & purificación , Calreticulina/metabolismo , Fibrinolisina/metabolismo , Plasminógeno/genética , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Dominios Proteicos/genética , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas de Inactivación de Genes , Línea Celular Tumoral , Neoplasias/fisiopatología
5.
FEBS Lett ; 596(11): 1401-1411, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35466397

RESUMEN

Amyloid-ß42 (Aß42) peptides are central to the amyloid pathology in Alzheimer's disease (AD). As biological mimetics, properties of synthetic Aß peptides usually vary between vendors and batches, thus impacting the reproducibility of experimental studies. Here, we tested recombinantly expressed Aß42 (Asp1 to Ala42) against synthetic Aß42 from different suppliers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), circular dichroism (CD) spectroscopy, thioflavin T aggregation, surface plasmon resonance, and MTT cell viability assays. Overall, our recombinant Aß42 provided a reproducible mimetic of desired properties. Across experimental approaches, the combined detection of Aß42 dimers and random coil to ß-sheet transition only correlated with aggregation-prone and cytotoxic peptides. Conclusively, combining MALDI-MS with CD appears to provide a rapid, reliable means to predict the 'bioactivity' of Aß42.


Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Humanos , Fragmentos de Péptidos/química , Reproducibilidad de los Resultados
6.
N Biotechnol ; 70: 28-38, 2022 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-35405333

RESUMEN

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required.


Asunto(s)
Esterasas , Polisacáridos , Aspergillus niger , Esterasas/química , Oligosacáridos/química , Filogenia , Polisacáridos/metabolismo , Especificidad por Sustrato
7.
J Biol Chem ; 298(1): 101483, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34896396

RESUMEN

We have previously developed a unique 8-amino acid Aß42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aß42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aß42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aß42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease.


Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Encéfalo , Fragmentos de Péptidos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/farmacocinética , Péptidos beta-Amiloides/farmacología , Animales , Encéfalo/metabolismo , Femenino , Masculino , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/farmacología
8.
JCI Insight ; 5(18)2020 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-32853178

RESUMEN

Based on its clinical benefits, Trikafta - the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) - was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661 plus VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to approximately 62% of WT CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K, and N1303K), confined to MSD1, MSD2, NBD1, and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.


Asunto(s)
Aminofenoles/farmacología , Benzodioxoles/farmacología , Bronquios/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Indoles/farmacología , Mutación , Pliegue de Proteína , Pirazoles/farmacología , Piridinas/farmacología , Quinolinas/farmacología , Bronquios/metabolismo , Bronquios/patología , Células Cultivadas , Fibrosis Quística/genética , Fibrosis Quística/patología , Combinación de Medicamentos , Humanos
9.
Mol Pharmacol ; 98(1): 1-12, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32362584

RESUMEN

Glial cell line-derived neurotrophic factor (GDNF) binds the GFRα1 receptor, and the GDNF-GFRα1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFRα1-RET signaling complex, agents that bind and activate RET directly and independently of GFRα1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFRα1 coexpression. However, RET activation by these ligands is constrained by GFRα1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT: A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor α, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.


Asunto(s)
Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Naftalenosulfonatos/administración & dosificación , Proteínas Proto-Oncogénicas c-ret/química , Proteínas Proto-Oncogénicas c-ret/metabolismo , Retinitis Pigmentosa/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Ligandos , Ratones , Naftalenosulfonatos/farmacología , Dominios Proteicos , Proteínas Proto-Oncogénicas c-ret/agonistas , Retinitis Pigmentosa/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/administración & dosificación
10.
Mol Cell Biol ; 40(15)2020 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-32366382

RESUMEN

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9 and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here, we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps the pSpt5-binding site and that can interact with single-stranded nucleic acid or with the polymerase-associated factor (PAF) complex in vitro We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Factores de Elongación Transcripcional/genética , Fosforilación , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética/genética , Factores de Elongación Transcripcional/metabolismo
11.
Science ; 368(6486)2020 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-32241924

RESUMEN

The success of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors (PARPi) to treat cancer relates to their ability to trap PARP-1 at the site of a DNA break. Although different forms of PARPi all target the catalytic center of the enzyme, they have variable abilities to trap PARP-1. We found that several structurally distinct PARPi drive PARP-1 allostery to promote release from a DNA break. Other inhibitors drive allostery to retain PARP-1 on a DNA break. Further, we generated a new PARPi compound, converting an allosteric pro-release compound to a pro-retention compound and increasing its ability to kill cancer cells. These developments are pertinent to clinical applications where PARP-1 trapping is either desirable or undesirable.


Asunto(s)
Regulación Alostérica/efectos de los fármacos , Roturas del ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Neoplasias/enzimología , Poli(ADP-Ribosa) Polimerasa-1/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Bencimidazoles/química , Bencimidazoles/farmacología , Línea Celular Tumoral , Humanos , Isoindoles/química , Isoindoles/farmacología , Piperazinas/química , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Dominios Proteicos
12.
Nat Commun ; 10(1): 2240, 2019 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-31110178

RESUMEN

The beta-site APP cleaving enzyme 1 (BACE1) is known primarily for its initial cleavage of the amyloid precursor protein (APP), which ultimately leads to the generation of Aß peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aß40 and Aß42 into a common Aß34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aß34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aß34 levels correlate with the overall Aß clearance rates in amyloid positive individuals. Using CSF samples from the PREVENT-AD cohort (cognitively normal individuals at risk for Alzheimer's disease), we further demonstrate that the Aß34/Aß42 ratio, representing Aß degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aß34 represents a marker of amyloid clearance and may be helpful for the characterization of Aß turnover in clinical samples.


Asunto(s)
Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Disfunción Cognitiva/patología , Fragmentos de Péptidos/metabolismo , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Ácido Aspártico Endopeptidasas/genética , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Disfunción Cognitiva/líquido cefalorraquídeo , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Fragmentos de Péptidos/líquido cefalorraquídeo , Proteolisis , Ratas , Ratas Sprague-Dawley
13.
J Neurochem ; 150(1): 74-87, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31077378

RESUMEN

Soluble oligomers of the 42-amino acid amyloid beta (Aß42) peptide are highly toxic and suspected as the causative agent of synaptic dysfunction and neuronal loss in Alzheimer's disease (AD). Previously, we have shown that a small, D-amino acid Aß42-oligomer interacting peptide (D-AIP) can neutralize human Aß42-mediated toxicity using in vitro and cell-based assays. In the present longitudinal study using a transgenic Drosophila melanogaster model, advanced live confocal imaging and mass spectrometry imaging (MALDI-MSI) showed that the eight amino acid D-AIP can attenuate Aß42-induced toxicity in vivo. By separating male and female flies into distinct groups, the resultant distribution of ingested D-AIP was different between the sexes. The Aß42-induced 'rough eye' phenotype could be rescued in the female transgenics, likely because of the co-localization of D-AIP with human Aß42 in the female fly heads. Interestingly, the phenotype could not be rescued in the male transgenics, likely because of the co-localization of D-AIP with a confounding male-specific sex peptide (Acp70A candidate in MSI spectra) in the gut of the male flies. As a novel, more cost-effective strategy to prevent toxic amyloid formation during the early stages of AD (i.e. neutralization of toxic low-order Aß42 oligomers without creating larger aggregates in the process), our longitudinal study establishes that D-AIP is a stable and highly effective neutralizer of toxic Aß42 peptides in vivo. Cover Image for this issue: doi: 10.1111/jnc.14512.


Asunto(s)
Péptidos beta-Amiloides/efectos de los fármacos , Péptidos beta-Amiloides/toxicidad , Encéfalo/efectos de los fármacos , Péptidos/farmacología , Animales , Animales Modificados Genéticamente , Drosophila melanogaster , Femenino , Humanos , Estudios Longitudinales , Masculino
14.
J Alzheimers Dis ; 69(2): 463-478, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31006686

RESUMEN

There is increasing evidence suggesting that amyloidogenic proteins might form deposits in non-neuronal tissues in neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. However, the detection of these aggregation-prone proteins within the human skin has been controversial. Using immunohistochemistry (IHC) and mass spectrometry tissue imaging (MALDI-MSI), fresh frozen human skin samples were analyzed for the expression and localization of neurodegenerative disease-related proteins. While α-synuclein was detected throughout the epidermal layer of the auricular samples (IHC and MALDI-MSI), tau and Aß34 were also localized to the epidermal layer (IHC). In addition to Aß peptides of varying length (e.g., Aß40, Aß42, Aß34), we also were able to detect inflammatory markers within the same sample sets (e.g., thymosin ß-4, psoriasin). While previous literature has described α-synuclein in the nucleus of neurons (e.g., Parkinson's disease), our current detection of α-synuclein in the nucleus of skin cells is novel. Imaging of α-synuclein or tau revealed that their presence was similar between the young and old samples in our present study. Future work may reveal differences relevant for diagnosis between these proteins at the molecular level (e.g., age-dependent post-translational modifications). Our novel detection of Aß34 in human skin suggests that, just like in the brain, it may represent a stable intermediate of the Aß40 and Aß42 degradation pathway.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Epidermis/metabolismo , Mediadores de Inflamación/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Fragmentos de Péptidos/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Anciano , Péptidos beta-Amiloides/análisis , Niño , Epidermis/química , Epidermis/patología , Femenino , Humanos , Mediadores de Inflamación/análisis , Masculino , Persona de Mediana Edad , Enfermedades Neurodegenerativas/patología , Fragmentos de Péptidos/análisis , Piel/química , Piel/metabolismo , Piel/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , alfa-Sinucleína/análisis , Proteínas tau/análisis
15.
Nat Med ; 24(11): 1732-1742, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30297908

RESUMEN

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50-100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Fibrosis Quística/tratamiento farmacológico , Pliegue de Proteína/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Regulación Alostérica/efectos de los fármacos , Bronquios/citología , Bronquios/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mutación , Mucosa Nasal/citología , Mucosa Nasal/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad
16.
J Immunol ; 200(4): 1471-1479, 2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29335257

RESUMEN

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein-coupled receptor (GPCR) leading to PLCß activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.


Asunto(s)
Enterotoxinas/inmunología , Laminina/inmunología , Activación de Linfocitos/inmunología , Infecciones Estafilocócicas/inmunología , Linfocitos T/inmunología , Animales , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Choque Séptico/inmunología , Staphylococcus aureus/inmunología , Superantígenos/inmunología
17.
ACS Chem Neurosci ; 9(2): 260-271, 2018 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-29078046

RESUMEN

Dendritic polyglycerols (dPG), particularly dendritic polyglycerol sulfates (dPGS), have been intensively studied due to their intrinsic anti-inflammatory activity. As related to brain pathologies involving neuroinflammation, the current study examined if dPG and dPGS can (i) regulate neuroglial activation, and (ii) normalize the morphology and function of excitatory postsynaptic dendritic spines adversely affected by the neurotoxic 42 amino acid amyloid-ß (Aß42) peptide of Alzheimer disease (AD). The exact role of neuroglia, such as microglia and astrocytes, remains controversial especially their positive and negative impact on inflammatory processes in AD. To test dPGS effectiveness in AD models we used primary neuroglia and organotypic hippocampal slice cultures exposed to Aß42 peptide. Overall, our data indicate that dPGS is taken up by both microglia and astrocytes in a concentration- and time-dependent manner. The mechanism of action of dPGS involves binding to Aß42, i.e., a direct interaction between dPGS and Aß42 species interfered with Aß fibril formation and reduced the production of the neuroinflammagen lipocalin-2 (LCN2) mainly in astrocytes. Moreover, dPGS normalized the impairment of neuroglia and prevented the loss of dendritic spines at excitatory synapses in the hippocampus. In summary, dPGS has desirable therapeutic properties that may help reduce amyloid-induced neuroinflammation and neurotoxicity in AD.


Asunto(s)
Dendrímeros/farmacología , Espinas Dendríticas/efectos de los fármacos , Glicerol/análogos & derivados , Glicerol/farmacología , Neuroglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Sinapsis/efectos de los fármacos , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Lipocalina 2/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Neuroglía/metabolismo , Neuroglía/patología , Neuroinmunomodulación/efectos de los fármacos , Neuroinmunomodulación/fisiología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Resonancia por Plasmón de Superficie , Sinapsis/metabolismo , Sinapsis/patología , Técnicas de Cultivo de Tejidos
18.
J Am Chem Soc ; 139(21): 7355-7362, 2017 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-28475327

RESUMEN

The development of nucleic acid therapeutics has been hampered by issues associated with their stability and in vivo delivery. To address these challenges, we describe a new strategy to engineer DNA structures with strong binding affinity to human serum albumin (HSA). HSA is the most abundant protein in the blood and has a long circulation half-life (19 days). It has been shown to hinder phagocytosis, is retained in tumors, and aids in cellular penetration. Indeed, HSA has already been successfully used for the delivery of small-molecule drugs and nanoparticles. We show that conjugating dendritic alkyl chains to DNA creates amphiphiles that exhibit high-affinity (Kd in low nanomolar range) binding to HSA. Notably, complexation with HSA did not hinder the activity of silencing oligonucleotides inside cells, and the degradation of DNA strands in serum was significantly slowed. We also show that, in a site-specific manner, altering the number and orientation of the amphiphilic ligand on a self-assembled DNA nanocube can modulate the affinity of the DNA cage to HSA. Moreover, the serum half-life of the amphiphile bound to the cage and the protein was shown to reach up to 22 hours, whereas unconjugated single-stranded DNA was degraded within minutes. Therefore, adding protein-specific binding domains to DNA nanostructures can be used to rationally control the interface between synthetic nanostructures and biological systems. A major challenge with nanoparticles delivery is the quick formation of a protein corona (i.e., protein adsorbed on the nanoparticle surface) upon injection to biological media. We foresee such DNA cage-protein complexes as new tools to study the role of this protein adsorption layer with important implications in the efficient delivery of RNAi therapeutics in vitro and in vivo.


Asunto(s)
ADN/química , Nanoestructuras/química , Albúmina Sérica Humana/química , Sitios de Unión , Humanos , Modelos Moleculares
19.
Sci Transl Med ; 8(365): 365ra159, 2016 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-27856798

RESUMEN

Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.


Asunto(s)
Antineoplásicos/farmacología , Lactamas Macrocíclicas/farmacología , Microtúbulos/efectos de los fármacos , Neuronas/efectos de los fármacos , Oxazoles/farmacología , Alcaloides de la Vinca/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Cristalografía por Rayos X , Dimerización , Genómica , Humanos , Lactamas Macrocíclicas/química , Ratones , Microscopía Electrónica , Mitosis , Trasplante de Neoplasias , Oxazoles/química , Tubulina (Proteína)/química , Vinblastina/análogos & derivados , Vinblastina/química , Vinblastina/farmacología , Alcaloides de la Vinca/química , Vinorelbina
20.
J Biol Chem ; 291(45): 23817-23829, 2016 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-27634044

RESUMEN

Gram-negative bacteria use type IV secretion systems (T4SSs) for a variety of macromolecular transport processes that include the exchange of genetic material. The pKM101 plasmid encodes a T4SS similar to the well-studied model systems from Agrobacterium tumefaciens and Brucella suis Here, we studied the structure and function of TraE, a homolog of VirB8 that is an essential component of all T4SSs. Analysis by X-ray crystallography revealed a structure that is similar to other VirB8 homologs but displayed an altered dimerization interface. The dimerization interface observed in the X-ray structure was corroborated using the bacterial two-hybrid assay, biochemical characterization of the purified protein, and in vivo complementation, demonstrating that there are different modes of dimerization among VirB8 homologs. Analysis of interactions using the bacterial two-hybrid and cross-linking assays showed that TraE and its homologs from Agrobacterium, Brucella, and Helicobacter pylori form heterodimers. They also interact with heterologous VirB10 proteins, indicating a significant degree of plasticity in the protein-protein interactions of VirB8-like proteins. To further assess common features of VirB8-like proteins, we tested a series of small molecules derived from inhibitors of Brucella VirB8 dimerization. These molecules bound to TraE in vitro, docking predicted that they bind to a structurally conserved surface groove of the protein, and some of them inhibited pKM101 plasmid transfer. VirB8-like proteins thus share functionally important sites, and these can be exploited for the design of specific inhibitors of T4SS function.


Asunto(s)
Proteínas Bacterianas/química , Bacterias Gramnegativas/química , Plásmidos/química , Sistemas de Secreción Tipo IV/química , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Brucella suis/química , Brucella suis/metabolismo , Cristalografía por Rayos X , Bacterias Gramnegativas/metabolismo , Helicobacter pylori/química , Helicobacter pylori/metabolismo , Modelos Moleculares , Plásmidos/antagonistas & inhibidores , Plásmidos/metabolismo , Conformación Proteica , Mapas de Interacción de Proteínas , Multimerización de Proteína , Bibliotecas de Moléculas Pequeñas/farmacología , Sistemas de Secreción Tipo IV/antagonistas & inhibidores , Sistemas de Secreción Tipo IV/metabolismo
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