Результаты поиска

Isolation and characterization of binding proteins for retinol from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens.

Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of th…

Structure-Dependent Effects of Lipid Conjugation on Cytosolic Accumulation in Escherichia coli and Mammalian Cells.

Lipidation is a widely used strategy to promote membrane permeation, yet whether the factors governing lipid-driven accumulation are shared across mammalian and Gram-negative envelopes remains unresolved. Here, we apply the chloroalkane azide-based membrane penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. …

Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide.

Background: Intracellular delivery of high-molecular-weight proteins is limited by the cell membrane. Cell-penetrating peptides (CPPs) offer a potential solution, but effective cytosolic delivery remains hindered by endosomal sequestration. Pas2r12, a CPP-derived peptide, facilitates cytosolic delivery of proteins including immunoglobulin G. Because Pas2r12 internalization occurs via caveolae-dependent endocytosis, we hypothesized that cell-surface receptors contribute to uptake. Methods: HEK293 cells were treated with Pas2r12 alone or complexed with enhanced green fluorescent protein (EGFP). …

A novel genetically encoded indicator for deciphering cytosolic and mitochondrial nitric oxide in live cells.

Nitric oxide (NO) has been highlighted as a key gaseous signaling molecule in the body, playing a central role in various physiological and pathological processes. However, a comprehensive analysis of NO metabolism dynamics in living cells remains a significant challenge. To address this, we have developed and characterized a novel genetically encoded NO fluorescence sensor, GefiNO, to investigate NO metabolism dynamics in living cells and subcellular organelles. In the study, we described the protein detection capabilities and subcellular spatiotemporal localization of GefiNO sensors. By anal

Control of Replication Stress Response by Cytosolic Fe-S Cluster Assembly (CIA) Machinery.

Accurate DNA replication is essential for the maintenance of genome stability and the generation of healthy offspring. When DNA replication … lication stress. These clusters are assembled and delivered to target proteins that function in the cytosol and nucleus via the conserved cytosolic Fe-S cluster assembly (CIA) machinery and the CIA targeting complex. This review summarizes recent advances in understanding the structure and function of the CIA machinery in yeast and mammals, emphasizing th …

Split-luciferin Assay for Real-time Measurement of Cytosolic Molecular Accumulation in Live Mycobacteria.

Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant Mycobacterium tuberculosis strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacterium's complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumu…

Methylation of ciliary dynein motors involves the essential cytosolic assembly factor DNAAF3/PF22.

Axonemal dynein motors drive ciliary motility and can consist of up to twenty distinct components with a combined mass of ~2 MDa. In mammals, failure of dyneins to assemble within the axonemal superstructure leads to primary ciliary dyskinesia. Syndromic phenotypes include infertility, rhinitis, severe bronchial conditions, and situs inversus. Nineteen specific cytosolic factors (Dynein Axonemal Assembly Factors; DNAAFs) are necessary for axonemal dynein assembly, although the detailed mechanisms involved remain very unclear. Here, we identify the essential assembly factor DNAAF3 as a structur…

Protective role of cytosolic prion protein against virus infection in prion-infected cells.

UNLABELLED: Production of the amyloidogenic prion protein, PrPSc, which forms infectious protein aggregates, or prions, is a key pathogenic … necroptosis. We also found that prion-infected cells accumulated a portion of PrP molecules in the cytosol, and that the N-terminal potential nuclear translocation signal of PrP impeded NF-κB nuclear translocation. These results suggest that PrPSc might play a functional role in protection against viral infections by stimulating the NLRP3 inflammasome-de …

The Salmonella virulence protein PagN contributes to the advent of a hyper-replicating cytosolic bacterial population.

Salmonella enterica subspecies enterica serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, Salmonella is enclosed within a membrane-bound vacuole known as a Salmonella-containing vacuole (SCV). However, a subset of Salmonella has the capability to prematurely rupture the SCV and escape, resulting in Salmonella hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights pagN vacuolar upregulation. Here,…

The cytosolic DNA sensor AIM2 promotes Helicobacter-induced gastric pathology via the inflammasome.

Helicobacter pylori (H. pylori) infection can trigger chronic gastric inflammation perpetuated by overactivation of the innate immune system, leading to a cascade of precancerous lesions culminating in gastric cancer. However, key regulators of innate immunity that promote H. pylori-induced gastric pathology remain ill-defined. The innate immune cytosolic DNA sensor absent in melanoma 2 (AIM2) contributes to the pathogenesis of numerous autoimmune and chronic inflammatory diseases, as well as cancers including gastric cancer. We therefore investigated whether AIM2 contributed to the pathogenes…

The Plasma Membrane Ca2+-ATPase, a Molecular Target for Tau-induced Cytosolic Calcium Dysregulation.

Disruption of calcium (Ca2+) homeostasis is emerging as a prevalent feature of aging and aging-associated neurodegenerative diseases, including Alzheimer's disease (AD), the most common type of tauopathy. This disease is characterized by the combined presence of extracellular neuritic plaques composed by amyloid β-peptides (Aβ) and neurofibrillary tangles of tau. The association of calcium dyshomeostasis with Aβ has been extensively studied, however its link with tau has been less investigated. Thus, this review will concentrate on the functional link between tau and the plasma membrane Ca2+ p

Оптимизация анализа насыщенности БРЕТ для надежных и чувствительных исследований взаимодействия цитосолика и протеина.

Насыщение биолюминесцентным резонансом передачи энергии (BRET) является методом изучения взаимодействия белка и протеина (PPI) при количественной оценке зависимости сигнала BRET от отношения к выражению приемщика/донора (A:D). В этом исследовании, используя очень яркую пару Nluc/YPF BRET, приобретенную соответственно с помощью микрозвукового считывателя и автоматизированной конфокальной микроскопии, мы значительно улучшили анализ насыщенности BRET путем расширения диапазона обнаружения выражения A:D и нормализации выражения A:D с помощью нового зонда, свободного от BRET. Далее мы обнаружили, ч

Совпадение в аннотации оригинала
Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein-protein interaction (PPI) upon quantification of … ing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabie …

Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins.

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistenc … ered a role for the host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host E …

Clinical utility of anti-cytosolic 5'-nucleotidase 1A antibody in idiopathic inflammatory myopathies.

OBJECTIVE: To define the clinicopathologic features and diagnostic utility associated with anti-cytosolic 5'-nucleotidase 1A (NT5C1A) antibody seropositivity in idiopathic inflammatory myopathies (IIMs). METHODS: Anti-NT5C1A antibody status was clinically tested between 2014 and 2019 in the Washington University neuromuscular clinical laboratory. Using clinicopathologic information available for 593 patients, we classified them as inclusion body myositis (IBM), dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy (IMNM), nonspecific myositis, or noninflammatory muscl…

A key cytosolic iron-sulfur cluster synthesis protein localizes to the mitochondrion of Toxoplasma gondii.

Iron-sulfur (Fe-S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe-S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe-S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe-S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe-S clusters assemble, and has a cytosolic loca…

Melatonin inhibits cytosolic mitochondrial DNA-induced neuroinflammatory signaling in accelerated aging and neurodegeneration.

Chronic inflammation is a pathologic feature of neurodegeneration and aging; however, the mechanism regulating this process is not understood. Melatonin, an endogenous free radical scavenger synthesized by neuronal mitochondria, decreases with aging and neurodegeneration. We proposed that insufficient melatonin levels impair mitochondrial homeostasis, resulting in mitochondrial DNA (mtDNA) release and activation of cytosolic DNA-mediated inflammatory response in neurons. We found increased mitochondrial oxidative stress and decreased mitochondrial membrane potential, with higher mtDNA release …

Linkage of typically cytosolic peroxidases to erythrocyte membrane - A possible mechanism of protection in Hereditary Spherocytosis.

Hereditary Spherocytosis (HS) is a non-immune hemolytic anemia associated to oxidative stress (OS), namely to the linkage of cytosolic antioxidant enzymes to the erythrocyte membrane. Our aims were to evaluate erythrocyte OS changes and the membrane linkage of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) in unsplenectomized (unspl) and splenectomized (spl) HS patients and to search for associations with clinical severity (in unspl HS patients). We studied 114 HS patients (74 unspl and 40 spl) and 30 healthy individuals and we evaluated membrane bound hemoglobin (MBH)…

Autoantibodies to Cytosolic 5'-Nucleotidase 1A in Primary Sjögren's Syndrome and Systemic Lupus Erythematosus.

INTRODUCTION: Autoantibodies to cytosolic 5'-nucleotidase 1A (cN-1A; NT5C1A) have a high specificity when differentiating sporadic inclusion body myositis from polymyositis and dermatomyositis. In primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE) anti-cN-1A autoantibodies can be detected as well. However, various frequencies of anti-cN-1A reactivity have been reported in SLE and pSS, which may at least in part be explained by the different assays used. Here, we determined the occurrence of anti-cN-1A reactivity in a large number of patients with pSS and SLE using one stan…

Pathomechanisms of anti-cytosolic 5'-nucleotidase 1A autoantibodies in sporadic inclusion body myositis.

OBJECTIVE: Sporadic inclusion body myositis (sIBM), an intractable progressive muscle disease, frequently occurs in older persons. sIBM pathogenesis may involve protein degradation dysfunction and immune abnormalities. Autoantibodies recognizing cytosolic 5'-nucleotidase 1A (cN1A) were found in plasma and serum from sIBM patients. However, whether anti-cN1A autoantibodies play a pathogenic role in sIBM is controversial. This study investigated the pathogenic properties of anti-cN1A autoantibodies in sIBM pathogenesis. METHODS: We developed a cell-based assay to detect anti-cN1A autoantibodies…