RESUMO
Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.
Assuntos
Cicatriz/metabolismo , Colágeno Tipo V/deficiência , Colágeno Tipo V/metabolismo , Traumatismos Cardíacos/metabolismo , Contração Miocárdica/genética , Miofibroblastos/metabolismo , Animais , Cicatriz/genética , Cicatriz/fisiopatologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo V/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica/genética , Integrinas/antagonistas & inibidores , Integrinas/genética , Integrinas/metabolismo , Isoproterenol/farmacologia , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Força Atômica/instrumentação , Microscopia Eletrônica de Transmissão , Contração Miocárdica/efeitos dos fármacos , Miofibroblastos/citologia , Miofibroblastos/patologia , Miofibroblastos/ultraestrutura , Análise de Componente Principal , Proteômica , RNA-Seq , Análise de Célula ÚnicaRESUMO
Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.
Assuntos
Adipócitos , Proteínas de Ligação ao Cálcio , Metabolismo dos Lipídeos , Proteínas de Membrana , Animais , Feminino , Humanos , Camundongos , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Placenta , Triglicerídeos/metabolismo , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Ácidos Graxos/metabolismo , Hipotermia/metabolismo , TermogêneseRESUMO
Brain rhythms provide the timing for recruitment of brain activity required for linking together neuronal ensembles engaged in specific tasks. The γ-oscillations (30 to 120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here, we report on a potent brain-permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a class of therapeutics for AD. We employed anatomical, in vitro and in vivo electrophysiological, and behavioral methods to examine the effects of our lead therapeutic candidate small molecule. As a novel in central nervous system pharmacotherapy, our lead molecule acts as a potent, efficacious, and selective negative allosteric modulator of the γ-aminobutyric acid type A receptors most likely assembled from α1ß2δ subunits. These receptors, identified through anatomical and pharmacological means, underlie the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. When orally administered twice daily for 2 wk, DDL-920 restored the cognitive/memory impairments of 3- to 4-mo-old AD model mice as measured by their performance in the Barnes maze. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.
Assuntos
Doença de Alzheimer , Cognição , Modelos Animais de Doenças , Ritmo Gama , Animais , Doença de Alzheimer/tratamento farmacológico , Camundongos , Cognição/efeitos dos fármacos , Ritmo Gama/efeitos dos fármacos , Memória/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Camundongos Transgênicos , Humanos , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Alanina/análogos & derivados , AzepinasRESUMO
Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.
Assuntos
Cromossomos Humanos/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mitose , Cromossomos Humanos/genética , Fosfatases de Especificidade Dupla/genética , Células HCT116 , Células HeLa , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Mutação , Fosforilação/genéticaRESUMO
Fatigue and other deleterious mood alterations resulting from prolonged efforts such as a long work shift can lead to a decrease in vigilance and cognitive performance, increasing the likelihood of errors during the execution of attention-demanding activities such as piloting an aircraft or performing medical procedures. Thus, a method to rapidly and objectively assess the risk for such cognitive fatigue would be of value. The objective of the study was the identification in saliva-borne exosomes of molecular signals associated with changes in mood and fatigue that may increase the risk of reduced cognitive performance. Using integrated multiomics analysis of exosomes from the saliva of medical residents before and after a 12 h work shift, we observed changes in the abundances of several proteins and miRNAs that were associated with various mood states, and specifically fatigue, as determined by a Profile of Mood States questionnaire. The findings herein point to a promising protein biomarker, phosphoglycerate kinase 1 (PGK1), that was associated with fatigue and displayed changes in abundance in saliva, and we suggest a possible biological mechanism whereby the expression of the PGK1 gene is regulated by miR3185 in response to fatigue. Overall, these data suggest that multiomics analysis of salivary exosomes has merit for identifying novel biomarkers associated with changes in mood states and fatigue. The promising biomarker protein presents an opportunity for the development of a rapid saliva-based test for the assessment of these changes.
Assuntos
Exossomos , MicroRNAs , Biomarcadores/metabolismo , Exossomos/genética , Exossomos/metabolismo , MicroRNAs/metabolismo , Saliva/metabolismoRESUMO
Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutant TTR gene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR.
Assuntos
Amiloide/química , Miocárdio/química , Pré-Albumina/química , Amiloide/metabolismo , Neuropatias Amiloides Familiares/metabolismo , Neuropatias Amiloides Familiares/patologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Pré-Albumina/metabolismoRESUMO
Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson's disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson's disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson's disease.
Assuntos
Homeostase , Ferro/metabolismo , Lactoferrina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sítios de Ligação , Células HEK293 , Humanos , Lactoferrina/química , Modelos Moleculares , Conformação ProteicaRESUMO
Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and ß-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and ß-subunits with ER chaperone BiP and impaired assembly of α/ß-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.
Assuntos
Retículo Endoplasmático/enzimologia , Células Epiteliais/enzimologia , Mucosa Gástrica/enzimologia , Gastrite/enzimologia , Proteínas de Choque Térmico/metabolismo , Infecções por Helicobacter/enzimologia , Helicobacter pylori/patogenicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Cultivadas , Retículo Endoplasmático/microbiologia , Chaperona BiP do Retículo Endoplasmático , Estabilidade Enzimática , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Gastrite/genética , Gastrite/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , ATPase Trocadora de Sódio-Potássio/genética , UbiquitinaçãoRESUMO
Oligomeric forms of α-synuclein are believed to cause mitochondrial injury, which may contribute to neurotoxicity in Parkinson's disease (PD). Here oligomers of α-synuclein were prepared using the dopamine metabolite, DOPAL (3,4-dihydroxyphenyl-acetaldehyde), in the presence of guanidinium hydrochloride. Electron microscopy, mass spectrometry, and Western blotting studies revealed enhanced and stable oligomerization with DOPAL compared with dopamine or CuCl2 /H2 O2 . Using isolated mouse brain mitochondria, DOPAL-oligomerized α-synuclein (DOS) significantly inhibited oxygen consumption rates compared with untreated, control-fibrillated, and dopamine-fibrillated synuclein, or with monomeric α-synuclein. Inhibition was greater in the presence of malate plus pyruvate than with succinate, suggesting the involvement of mitochondrial complex I. Mitochondrial membrane potential studies using fluorescent probes, JC-1, and Safranin O also detected enhanced inhibition by DOS compared with the other aggregated forms of α-synuclein. Testing a small customized chemical library, four compounds were identified that rescued membrane potential from DOS injury. While diverse in chemical structure and mechanism, each compound has been reported to interact with mitochondrial complex I. Western blotting studies revealed that none of the four compounds disrupted the oligomeric banding pattern of DOS, suggesting their protection involved direct mitochondrial interaction. The remaining set of chemicals also did not disrupt oligomeric banding, attesting to the high structural stability of this α-synuclein proteoform. DOPAL and α-synuclein are both found in dopaminergic neurons, where their levels are elevated in PD and in animal models exposed to chemical toxicants, including agricultural pesticides. The current study provides further evidence of α-synuclein-induced mitochondrial injury and a likely role in PD neuropathology.
Assuntos
Dopamina/metabolismo , Mitocôndrias/metabolismo , alfa-Sinucleína/metabolismo , Animais , Dopamina/química , Dopamina/farmacologia , Feminino , Masculino , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio , Doença de Parkinson , Agregados Proteicos/efeitos dos fármacos , alfa-Sinucleína/química , alfa-Sinucleína/farmacologia , alfa-Sinucleína/ultraestruturaRESUMO
The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.
Assuntos
Adenosina Trifosfatases/metabolismo , Microtúbulos/metabolismo , Proteômica/métodos , Adenosina Trifosfatases/química , Núcleo Celular/metabolismo , Células HeLa , Humanos , Katanina , Espectrometria de Massas , Meiose , Mapas de Interação de ProteínasRESUMO
Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation.
Assuntos
Chlamydomonas reinhardtii/enzimologia , Complexo Citocromos b6f/química , Complexos de Proteínas Captadores de Luz/química , Proteínas Serina-Treonina Quinases/química , Chlamydomonas reinhardtii/genética , Complexo Citocromos b6f/genética , Complexo Citocromos b6f/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Oxirredução , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
The aim of this pilot study is to evaluate and compare the quality of the genomics and proteomics data obtained from paired Formalin Fixed Paraffin Embedded (FFPE) and frozen (FF) tissue percutaneous core biopsies of Liver Imaging Reporting and Data System 5 (LIRADS 5) hepatocellular carcinoma (HCC) of varying histological grades. The preliminary data identified differentially expressed proteins and genes in poor, moderate and well differentiated HCC biopsies, with a greater efficacy in fresh frozen samples. The data offered valuable insights into the characteristics and suitability of samples for future studies.
RESUMO
Brain rhythms provide the timing and concurrence of brain activity required for linking together neuronal ensembles engaged in specific tasks. In particular, the γ-oscillations (30-120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here we report on a potent brain permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a new class of therapeutics for AD. As a first in CNS pharmacotherapy, our lead candidate acts as a potent, efficacious, and selective negative allosteric modulator (NAM) of the γ-aminobutyric acid type A receptors (GABA A Rs) assembled from α1ß2δ subunits. We identified these receptors through anatomical and pharmacological means to mediate the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.
RESUMO
The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virus-like particles (VLP) and VLP fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/metabolismo , Complexo de Golgi/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Transmembrane α-helical domains of membrane proteins tend to remain structured in the gas phase, presenting a challenge for efficient electron capture/transfer dissociation during top-down dissociation mass spectrometry (MS) experiments. In this study, we compare results from different dissociation modes on a modern Orbitrap platform applied to a model integral membrane protein containing two transmembrane helices, the c-subunit of the Fo domain of the chloroplast ATP synthase. Using commercially available options, we compare collisionally activated dissociation (CAD) with the related variant higher-energy collisional dissociation (HCD) and with electron transfer dissociation (ETD). HCD performed better than CAD and ETD. A combined method utilizing both ETD and HCD (EThcD) demonstrates significant synergy over HCD or ETD alone, representing a robust option analogous to activated ion electron capture dissociation, whereby an infrared laser was used to heat the protein ion alongside electron bombardment. Ultraviolet photodissociation at 213 nm displays at least three backbone dissociation mechanisms and covered nearly 100% of backbone bonds, suggesting significant potential for this technique.
Assuntos
ATPases de Cloroplastos Translocadoras de Prótons/química , Espectrometria de Massas/métodos , Proteínas de Membrana/química , ATPases de Cloroplastos Translocadoras de Prótons/isolamento & purificação , Transporte de Elétrons , Espectrometria de Massas/instrumentação , Proteínas de Membrana/isolamento & purificação , Processos Fotoquímicos , Conformação Proteica em alfa-Hélice , Raios UltravioletaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aß. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.
RESUMO
AIM: We have previously reported that cambinol (DDL-112), a known inhibitor of neutral sphingomyelinase-2 (nSMase2), suppressed extracellular vesicle (EV)/exosome production in vitro in a cell model and reduced tau seed propagation. The enzyme nSMase2 is involved in the production of exosomes carrying proteopathic seeds and could contribute to cell-to-cell transmission of pathological protein aggregates implicated in neurodegenerative diseases such as Parkinson's disease (PD). Here, we performed in vivo studies to determine if DDL-112 can reduce brain EV/exosome production and proteopathic alpha synuclein (αSyn) spread in a PD mouse model. METHODS: The acute effects of single-dose treatment with DDL-112 on interleukin-1ß-induced extracellular vesicle (EV) release in brain tissue of Thy1-αSyn PD model mice and chronic effects of 5 week DDL-112 treatment on behavioral/motor function and proteinase K-resistant αSyn aggregates in the PD model were determined. RESULTS/DISCUSSION: In the acute study, pre-treatment with DDL-112 reduced EV/exosome biogenesis and in the chronic study, treatment with DDL-112 was associated with a reduction in αSyn aggregates in the substantia nigra and improvement in motor function. Inhibition of nSMase2 thus offers a new approach to therapeutic development for neurodegenerative diseases with the potential to reduce the spread of disease-specific proteopathic proteins.
Assuntos
Encéfalo/metabolismo , Inibidores Enzimáticos/farmacologia , Exossomos/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , alfa-Sinucleína/metabolismo , Animais , Modelos Animais de Doenças , Exossomos/ultraestrutura , Camundongos Transgênicos , Naftalenos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Pirimidinonas/farmacologia , Sirtuínas/metabolismo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Carbon monoxide (CO)-releasing antibody conjugates were synthesized utilizing a photoactivatable CO-releasing molecule (photoCORM) and mouse monoclonal antibodies linked by a biotin-streptavidin system. Different monoclonal antibodies raised against different surface-expressed antigens that are implicated in ovarian cancer afforded a family of antibody-photoCORM conjugates (Ab-photoCORMs). In an immunosorbent/cell viability assay, Ab-photoCORMs accumulated onto ovarian cancer cells expressing the target antigens, delivering cytotoxic doses of CO in vitro. The results described here provide the first example of an "immunoCORM", a proof-of-the-concept antibody-drug conjugate that delivers a gaseous molecule as a warhead to ovarian cancer.
RESUMO
Several 'super-complexes' of individual hetero-oligomeric membrane protein complexes, whose function is to facilitate intra-membrane electron and proton transfer and harvesting of light energy, have been previously characterized in the mitochondrial cristae and chloroplast thylakoid membranes. We report the presence of an intra-membrane super-complex dominated by the ATP-synthase, photosystem I (PSI) reaction-center complex and the ferredoxin-NADP+ Reductase (FNR) in the thylakoid membrane. The presence of the super-complex has been documented by mass spectrometry, clear-native PAGE and Western Blot analyses. This is the first documented presence of ATP synthase in a super-complex with the PSI reaction-center located in the non-appressed stromal domain of the thylakoid membrane.
Assuntos
Cloroplastos/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Óxido Nítrico Sintase/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tilacoides/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte de Elétrons , Fotossíntese , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Spinacia oleracea/crescimento & desenvolvimento , Spinacia oleracea/metabolismoRESUMO
We report the discovery of a novel class of compounds that function as dual inhibitors of the enzymes neutral sphingomyelinase-2 (nSMase2) and acetylcholinesterase (AChE). Inhibition of these enzymes provides a unique strategy to suppress the propagation of tau pathology in the treatment of Alzheimer's disease (AD). We describe the key SAR elements that affect relative nSMase2 and/or AChE inhibitor effects and potency, in addition to the identification of two analogs that suppress the release of tau-bearing exosomes in vitro and in vivo. Identification of these novel dual nSMase2/AChE inhibitors represents a new therapeutic approach to AD and has the potential to lead to the development of truly disease-modifying therapeutics.