ABSTRACT
Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition.Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN-induced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1.
Subject(s)
Mitophagy , Parkinson Disease , Protein Kinases/metabolism , X-Box Binding Protein 1/metabolism , Animals , Autophagy , Endoribonucleases , Mice , Mitophagy/genetics , Parkinson Disease/genetics , Phosphorylation , Protein Serine-Threonine KinasesABSTRACT
The enzyme tyrosinase is involved in the biosynthesis of melanin and the enzymatic browning of fruits and vegetables, and therefore, its inhibitors have potential to treat hyperpigmentary disorders or to function as food antibrowning agents. The use of hydrazine monohydrate as a reagent to prepare chemically engineered extracts can lead to semisynthetic compounds that contain the portion N-N, a fragment rarely found in natural products and present in some tyrosinase inhibitors. Here, we report the tyrosinase inhibition screening of a series of chemically engineered extracts that are diversified by reaction with hydrazine. LC-MS was used to evaluate the change in composition produced by the reaction. Bioguided fractionation of the most active chemically engineered extract, prepared from Matricaria recutita L., led to the discovery of a pyrazole that inhibits tyrosinase with an IC50 value of 28.20 ± 1.13 µM. This compound was produced by a one-pot double chemical transformation of its natural precursor, which includes an unexpected selective removal of one -OH group.
Subject(s)
Enzyme Inhibitors/chemistry , Hydrazines/chemistry , Matricaria/chemistry , Plant Extracts/chemistry , Chemical Engineering , Drug Design , Flavones/chemistry , Melanins/chemistry , Melanins/metabolism , Molecular Structure , Monophenol Monooxygenase/antagonists & inhibitors , Pyrazoles/chemistry , Structure-Activity RelationshipABSTRACT
INTRODUCTION: The PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. OBJECTIVE: To develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes. METHODS: The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. RESULTS: A bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system. CONCLUSION: A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Chromatography, Thin Layer/methods , Linoleic Acid/pharmacology , Plant Extracts/pharmacology , Salmonella typhimurium/drug effects , alpha-Linolenic Acid/pharmacology , Dimerization , Galactosides , Genes, Reporter , Hydrolysis , Indoles , Linoleic Acid/chemistry , Linoleic Acid/isolation & purification , Magnetic Resonance Spectroscopy , Magnoliopsida/chemistry , Mass Spectrometry , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Salmonella typhimurium/metabolism , Virulence , alpha-Linolenic Acid/chemistry , alpha-Linolenic Acid/isolation & purificationABSTRACT
Biological research and drug discovery critically depend on access to libraries of small molecules that have an affinity for biomacromolecules. By virtue of their sustained success as sources of lead compounds, natural products are recognized as "privileged" starting points in structural space for library development. Compared with synthetic compounds, natural products have distinguishing structural properties; indeed, researchers have begun to quantify and catalog the differences between the two classes of molecules. Measurable differences in the number of chiral centers, the degree of saturation, the presence of aromatic rings, and the number of the various heteroatoms are among the chief distinctions between natural and synthetic compounds. Natural products also include a significant proportion of recurring molecular scaffolds that are not present in currently marketed drugs: the bioactivity of these natural substructures has been refined over the long process of evolution. In this Account, we present our research aimed at preparing libraries of semisynthetic compounds, or chemically engineered extracts (CEEs), through chemical diversification of natural products mixtures. The approach relies on the power of numbers, that is, in the chemical alteration of a sizable fraction of the starting complex mixture. Major changes in composition can be achieved through the chemical transformation of reactive molecular fragments that are found in most natural products. If such fragments are common enough, their transformation represents an entry point for chemically altering a high proportion of the components of crude natural extracts. We have searched for common reactive fragments in the Dictionary of Natural Products (CRC Press) and identified several functional groups that are expected to be present in a large fraction of the components of an average natural crude extract. To date, we have used reactions that incorporate (i) nitrogen atoms through carbonyl groups, (ii) sulfur by transformation of -OH and amines, and (iii) bromine through double bonds and aromatic rings. The resulting CEEs had different composition and biomolecular properties than their natural progenitors. We isolated a semisynthetic ß-glucosidase inhibitor from a CEE prepared by reaction with benzenesulfonyl chloride, an antifungal pyrazole from a CEE prepared by reaction with hydrazine, and an acetylcholinesterase inhibitor from a CEE prepared through bromination. Our results illustrate how biological activity can be generated through chemical diversification of natural product mixtures. Moreover, the level of control that can be asserted in the process by judicious design and experimental choices underscores the potential for further development of CEEs in both basic research and drug discovery.
Subject(s)
Biological Products/chemistry , Chemical Engineering/methods , Drug Discovery/methods , Biological Products/metabolism , Humans , Plant Extracts/chemistry , Plant Extracts/metabolismABSTRACT
The chemical composition and the biomolecular properties of a crude plant extract were altered through bromination leading to the discovery of an acetylcholinesterase inhibitor.
Subject(s)
Bromine , Cholinesterase Inhibitors/chemical synthesis , Plant Extracts/chemistry , Argentina , Bromine/chemistry , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Halogenation , Magnetic Resonance Spectroscopy , Molecular StructureABSTRACT
A semisynthetic ß-glucosidase inhibitor was identified from a chemically engineered extract prepared by reaction with benzenesulfonyl chloride. The structure includes a natural histamine portion and a benzenesulfonyl portion introduced during the diversification step.
Subject(s)
Plant Extracts/chemistry , Sulfones/chemistry , beta-Glucosidase/antagonists & inhibitors , Chemical Engineering , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Urticaceae/chemistry , beta-Glucosidase/chemistryABSTRACT
The chemical composition and the biomolecular properties of a series of crude plant extracts were altered without previous knowledge of their detailed chemical composition.
Subject(s)
Plant Extracts/chemistry , Sulfones/chemistry , Glycoside Hydrolases/antagonists & inhibitors , Glycoside Hydrolases/metabolism , Phytotherapy , Plant Extracts/pharmacology , Plants, MedicinalABSTRACT
El presente estudio prospectivo, descriptivo reporta seis casos de absceso cerebral piógenos, atendidos en el Servicio de Medicina del Hospital II EsSalud de Cajamarca, Perú, durante el periodo de 1990 al 2006. La edad promedio fue de 42 años, la mínima 9 años y el 66 por ciento fueron mayores de 40 años; 3 casos de sexo masculino y 3 de sexo femenino. Los focos primarios fueron: bronquiectasia, otitis media crónica, absceso dentario, sinusitis crónica, de origen cardiogénico y en 1 caso no se determinó. La desnutrición, VIH positivo, diabetes mellitus y endocarditis bacteriana; en 2 no se determinó enfermedad de fondo y en 1 hubo curetaje de absceso dental. Los síntomas de inicio son: cefalea en 50 por ciento convulsiones 17 por ciento. El síntoma principal: convulsiones en 66 por ciento, la hemiparesia y alteraciones del sensorio. El cuadro clínico estuvo caracterizado por cefalea (83 por ciento) convulsiones (66 por ciento), hemiparesia (66 por ciento), fiebre (66 por ciento), afasia (50 por ciento) edema de papila y vómito (50 por ciento) de alteración de memoria, signos meningeos y signos prefrontales (33 por ciento). Los sindromes clínicos: sindrome de cefalea (33 por ciento), neurológico focal (83 por ciento) epileptico focal (67 por ciento) febril (67 por ciento) de HEC (50 por ciento), meníngeo (33por ciento) de funciones superiores (33 por ciento). La TAC y IRM cerebal reveló 6 casos de absceso cerebral, de localización hemisférica derecha en 5 casos, 3 únicos y 3 múltiples; 2 en el lóculo frontal, 1 frontapariental, 1 frontotemporal y 1 parietal. Dos casos en fase de cerebritis y 4 encapsulados, con edema circundante y efecto de masa. El tratamiento se realizó cocn cefotaxima 1gr c/6 hs. VEV/20 días. 1 caso se prolongó el tratamietnno hasta 60 días con metronidazol por vía oral. Las secuelas fueron: hemiparesia, alteraciones prefrontales, convulsiones, polineuropatía periférica por metronidazol. La evolución clínica y tomográfica fue favorable en ...
Subject(s)
Male , Female , Humans , Child , Adolescent , Adult , Middle Aged , Brain Abscess , Brain Abscess/diagnosis , Brain Abscess/epidemiology , Brain Abscess/therapy , Cefotaxime , Cefotaxime/administration & dosage , Clinical Diagnosis , Metronidazole , Metronidazole/administration & dosage , Epidemiology, Descriptive , Prospective StudiesABSTRACT
A new bioautographic assay suitable for the localisation of beta-glucosidase inhibitors present in a complex matrix is described. Enzyme activity was detected using esculin as the substrate to produce esculetin, which reacts with ferric ion to form a brown complex.