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Molecular Cell Dec 2023The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined...
The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.
Topics: Endoplasmic Reticulum; Proteins; Cytosol; RNA, Messenger; Protein Transport; Protein Biosynthesis
PubMed: 38134885
DOI: 10.1016/j.molcel.2023.11.025 -
Scientific Reports Aug 2020Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential...
Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential analysis of exosomal proteomes from cancerous and non-tumorigenic breast cell lines can provide valuable information related to breast cancer progression and metastasis. Moreover, such a comparison can be explored to find potentially new protein biomarkers for early disease detection. In this study, exosomal proteomes of MDA-MB-231, a metastatic breast cancer cell line, and MCF-10A, a non-cancerous epithelial breast cell line, were identified by nano-liquid chromatography coupled to tandem mass spectrometry. We also tested three exosomes isolation methods (ExoQuick, Ultracentrifugation (UC), and Ultrafiltration-Ultracentrifugation) and detergents (n-dodecyl β-D-maltoside, Triton X-100, and Digitonin) for solubilization of exosomal proteins and enhanced detection by mass spectrometry. A total of 1,107 exosomal proteins were identified in both cell lines, 726 of which were unique to the MDA-MB-231 breast cancer cell line. Among them, 87 proteins were predicted to be relevant to breast cancer and 16 proteins to cancer metastasis. Three exosomal membrane/surface proteins, glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), were identified as potential breast cancer biomarkers and validated with Western blotting and high-resolution flow cytometry. We demonstrated that exosomes are a rich source of breast cancer-related proteins and surface biomarkers that may be used for disease diagnosis and prognosis.
Topics: Biomarkers, Tumor; Breast Neoplasms; Exosomes; Female; Humans; Mass Spectrometry; Proteome; Proteomics; Tumor Cells, Cultured; Ultracentrifugation
PubMed: 32782317
DOI: 10.1038/s41598-020-70393-4 -
Redox Biology Jul 2023Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms...
Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms that regulate supercomplex abundance remain unclear. In this study, we examined the composition of supercomplexes derived from murine cardiac mitochondria and determined how their abundance changes with substrate provision or by genetically induced changes to the cardiac glucose-fatty acid cycle. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain constituents of Complexes I, III, IV, and V as well as accessory proteins involved in supercomplex assembly and stability, cristae architecture, carbohydrate and fat oxidation, and oxidant detoxification. Respiratory analysis of high molecular mass supercomplexes confirmed the presence of intact respirasomes, capable of transferring electrons from NADH to O. Provision of respiratory substrates to isolated mitochondria augmented supercomplex abundance, with fatty acyl substrate (octanoylcarnitine) promoting higher supercomplex abundance than carbohydrate-derived substrate (pyruvate). Mitochondria isolated from transgenic hearts that express kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Glyco), which decreases glucose utilization and increases reliance on fatty acid oxidation for energy, had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-expressing hearts (Glyco), the latter of which encourages reliance on glucose catabolism for energy. These findings indicate that high energetic reliance on fatty acid catabolism bolsters levels of mitochondrial supercomplexes, supporting the idea that the energetic state of the heart is regulatory factor in supercomplex assembly or stability.
Topics: Mice; Animals; Phosphofructokinase-2; Heart; Mitochondria, Heart; Glucose; Fatty Acids; Mammals
PubMed: 37210780
DOI: 10.1016/j.redox.2023.102740 -
The EMBO Journal Dec 2021Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting...
Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.
Topics: Animals; Cell Membrane; Mice; Mice, Inbred C57BL; Mice, Knockout; Necroptosis; Phosphorylation; Proteasome Endopeptidase Complex; Protein Kinases; Protein Multimerization; Ubiquitin Thiolesterase; Ubiquitination
PubMed: 34698396
DOI: 10.15252/embj.2019103718 -
Science Signaling Feb 2023Synaptotagmin-11 (Syt11) is a vesicle-trafficking protein that is linked genetically to Parkinson's disease (PD). Likewise, the protein α-synuclein regulates vesicle...
Synaptotagmin-11 (Syt11) is a vesicle-trafficking protein that is linked genetically to Parkinson's disease (PD). Likewise, the protein α-synuclein regulates vesicle trafficking, and its abnormal aggregation in neurons is the defining cytopathology of PD. Because of their functional similarities in the same disease context, we investigated whether the two proteins were connected. We found that Syt11 was palmitoylated in mouse and human brain tissue and in cultured cortical neurons and that this modification to Syt11 disrupted α-synuclein homeostasis in neurons. Palmitoylation of two cysteines adjacent to the transmembrane domain, Cys and Cys, localized Syt11 to digitonin-insoluble portions of intracellular membranes and protected it from degradation by the endolysosomal system. In neurons, palmitoylation of Syt11 increased its abundance and enhanced the binding of α-synuclein to intracellular membranes. As a result, the abundance of the physiologic tetrameric form of α-synuclein was decreased, and that of its aggregation-prone monomeric form was increased. These effects were replicated by overexpression of wild-type Syt11 but not a palmitoylation-deficient mutant. These findings suggest that palmitoylation-mediated increases in Syt11 amounts may promote pathological α-synuclein aggregation in PD.
Topics: Mice; Animals; Humans; Synaptotagmins; Parkinson Disease; alpha-Synuclein; Lipoylation; Neurons
PubMed: 36787382
DOI: 10.1126/scisignal.add7220 -
Drug Metabolism and Disposition: the... Dec 2022As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that...
As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that play a major role in drug metabolism and bioactivation of prodrugs have been detected in ocular tissues, but data on their quantitative expression is scarce. Also, many ophthalmic drugs are prone to hydrolysis. Metabolic characterization of individual ocular tissues is useful for the drug development process, and therefore, seven individual ocular tissues from human eyes were analyzed for the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC). Generic and selective human esterase substrates 4-nitrophenyl acetate (most esterases), D-luciferin methyl ester (CES1), fluorescein diacetate and procaine (CES2), and phenacetin (AADAC) were applied to determine the enzymes' specific activities. Enzyme kinetics and inhibition studies were performed with isoform-selective inhibitors digitonin (CES1) and verapamil and diltiazem (CES2). Enzyme contents were determined using quantitative targeted proteomics, and CES2 expression was confirmed by western blotting. The expression and activity of human CES1 among ocular tissues varied by >10-fold, with the highest levels found in the retina and iris-ciliary body. In contrast, human CES2 expression appeared lower and more similar between tissues, whereas AADAC could not be detected. Inhibition studies showed that hydrolysis of fluorescein diacetate is also catalyzed by enzymes other than CES2. This study provides, for the first time, quantitative information on the tissue-dependent expression of human ocular esterases, which can be useful for the development of ocular drugs, prodrugs, and in pharmacokinetic modeling of the eye. SIGNIFICANCE STATEMENT: Novel and comprehensive data on the protein expression and activities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical species, this study will improve the understanding of interspecies differences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.
Topics: Humans; Carboxylic Ester Hydrolases; Carboxylesterase; Prodrugs; Fluoresceins; Hydrolysis
PubMed: 36195336
DOI: 10.1124/dmd.122.000993 -
Scientific Reports Sep 2021Plasma membrane repair mechanisms are activated within seconds post-injury to promote rapid membrane resealing in eukaryotic cells and prevent cell death. However, less...
Plasma membrane repair mechanisms are activated within seconds post-injury to promote rapid membrane resealing in eukaryotic cells and prevent cell death. However, less is known about the regeneration phase that follows and how cells respond to injury in the short-term. Here, we provide a genome-wide study into the mRNA expression profile of MCF-7 breast cancer cells exposed to injury by digitonin, a mild non-ionic detergent that permeabilizes the plasma membrane. We focused on the early transcriptional signature and found a time-dependent increase in the number of differentially expressed (> twofold, P < 0.05) genes (34, 114 and 236 genes at 20-, 40- and 60-min post-injury, respectively). Pathway analysis highlighted a robust and gradual three-part transcriptional response: (1) prompt activation of immediate-early response genes, (2) activation of specific MAPK cascades and (3) induction of inflammatory and immune pathways. Therefore, plasma membrane injury triggers a rapid and strong stress and immunogenic response. Our meta-analysis suggests that this is a conserved transcriptome response to plasma membrane injury across different cell and injury types. Taken together, our study shows that injury has profound effects on the transcriptome of wounded cells in the regeneration phase (subsequent to membrane resealing), which is likely to influence cellular status and has been previously overlooked.
Topics: Animals; Cell Membrane; Computational Biology; Gene Expression Regulation; Humans; MAP Kinase Signaling System; MCF-7 Cells; RNA-Seq; Regeneration
PubMed: 34580330
DOI: 10.1038/s41598-021-98420-y -
Bioluminescent test systems based on firefly luciferase for studying stress effects on living cells.Biophysical Reviews Aug 2022The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly... (Review)
Review
The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly luciferase. The possibilities of using this reaction to study the influence of external effectors of a physical and chemical nature (temperature exposure, additions of drugs, membrane-active compounds, etc.) on living cells (prokaryotes and eukaryotes) are considered. Examples of the use of test systems based on living cells producing thermostable firefly luciferase for monitoring cellular homeostasis are given. The study of the kinetics of changes in the concentration of ATP and luciferase inside and outside cells made it possible to determine in dynamics the metabolic activity, cytotoxicity, and survival of cells under conditions of cellular stress, to study the processes of ATP synthesis/hydrolysis, and to evaluate the effectiveness of lytic agents in changing the permeability of the cell membrane.
PubMed: 36124280
DOI: 10.1007/s12551-022-00978-y -
Plant Methods Mar 2022Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid...
BACKGROUND
Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging.
RESULTS
Using a detergent mixture of 1% (w/V) n-dodecyl-β-D-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3-8% gel gradients for separation as methodological improvements in BN PAGE, several large photosystem (PS) I containing bands were detected. According to BN(/BN)/SDS PAGE and mass spectrometry analyses, these PSI bands proved to be PSI-NADH dehydrogenase-like megacomplexes more discernible in maize bundle sheath thylakoids, and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) more abundant in mesophyll thylakoids of lincomycin treated maize. For quantitative determination of the complexes and their comparison across taxa and physiological conditions, sample volumes applicable to the gel, correct baseline determination of the densitograms, evaluation methods to resolve complexes running together, calculation of their absolute/relative amounts and distribution among their different forms are proposed.
CONCLUSIONS
Here we report our experience in Blue/Clear-Native polyacrylamide gel electrophoretic separation of thylakoid complexes, their identification, quantitative determination and comparison in different samples. The applied conditions represent a powerful methodology for the analysis of thylakoid mega- and supercomplexes.
PubMed: 35241118
DOI: 10.1186/s13007-022-00858-2 -
Nature Communications Oct 2023Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite...
Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite intense investigations into their working mechanism and pharmaceutical potentials, how neurotransmitters activate these receptors remains unclear due to the lack of high-resolution structural information in the activated open state. Here we report near-atomic resolution structures resolved in digitonin consistent with all principle functional states of the human α1β GlyR, which is a major Cys-loop receptor that mediates inhibitory neurotransmission in the central nervous system of adults. Glycine binding induces cooperative and symmetric structural rearrangements in the neurotransmitter-binding extracellular domain but asymmetrical pore dilation in the transmembrane domain. Symmetric response in the extracellular domain is consistent with electrophysiological data showing cooperative glycine activation and contribution from both α1 and β subunits. A set of functionally essential but differentially charged amino acid residues in the transmembrane domain of the α1 and β subunits explains asymmetric activation. These findings provide a foundation for understanding how the gating of the Cys-loop receptor family members diverges to accommodate specific physiological environments.
Topics: Humans; Receptors, Glycine; Ion Channel Gating; Cysteine Loop Ligand-Gated Ion Channel Receptors; Synaptic Transmission; Glycine
PubMed: 37821459
DOI: 10.1038/s41467-023-42051-6