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Basic & Clinical Pharmacology &... Nov 2013Dermaseptin, an antimicrobial peptide participating in the host defence against pathogens, interacts with the membrane of target cells, leading to membrane...
Dermaseptin, an antimicrobial peptide participating in the host defence against pathogens, interacts with the membrane of target cells, leading to membrane permeabilization and eventual cell lysis. Dermaseptin has previously been shown to trigger haemolysis. Prior to haemolysis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase in cytosolic Ca²⁺ activity [(Ca²⁺)](i) and formation of ceramide. This study explored whether dermaseptin modifies [Ca²⁺](i) and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine exposure from annexin-V binding, haemolysis from haemoglobin release, ceramide formation from binding of fluorescent antibodies and [Ca²⁺](i) from Fluo3-fluorescence. A 48-hr exposure to dermaseptin (50 μM) was followed by a significant increase in [Ca²⁺](i), a significant increase ceramide abundance, a significant decrease in forward scatter and a significant increase in annexin-V binding. The annexin-V binding after dermaseptin treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. Dermaseptin triggers eryptosis, an effect at least partially due to entry of extracellular Ca²⁺.
Topics: Amphibian Proteins; Annexin A5; Antimicrobial Cationic Peptides; Apoptosis; Calcium; Cell Membrane; Cell Size; Ceramides; Cytosol; Erythrocytes; Hemolysis; Humans; Phosphatidylserines
PubMed: 23841716
DOI: 10.1111/bcpt.12096 -
Cell Jan 2009The ESCRT-III complex mediates membrane budding away from the cytosol in endosome biogenesis, cytokinesis, and viral budding. In this issue, Saksena et al. (2009) use an...
The ESCRT-III complex mediates membrane budding away from the cytosol in endosome biogenesis, cytokinesis, and viral budding. In this issue, Saksena et al. (2009) use an elegant fluorescence-based approach to define the sequential activation, recruitment, and disassembly of ESCRT-III subunits during membrane involution in vitro.
Topics: Cytosol; Endosomes; Humans; Spectrometry, Fluorescence; Vesicular Transport Proteins; Yeasts
PubMed: 19135881
DOI: 10.1016/j.cell.2008.12.029 -
Protein Science : a Publication of the... Aug 2001Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the...
Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria.
Topics: Aldehyde Dehydrogenase; Amino Acid Sequence; Cell-Free System; Centrifugation, Density Gradient; Cytosol; Enzyme Stability; Humans; Isoenzymes; Liver; Mitochondria; Molecular Sequence Data; Protein Denaturation; Protein Folding; Protein Precursors; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins; Trypsin; Urea; Yeasts
PubMed: 11468345
DOI: 10.1110/ps.5301 -
Critical Reviews in Biochemistry and... 2015A dedicated network of cellular factors ensures that proteins translocated into the endoplasmic reticulum (ER) are folded correctly before they exit this compartment en... (Review)
Review
A dedicated network of cellular factors ensures that proteins translocated into the endoplasmic reticulum (ER) are folded correctly before they exit this compartment en route to other cellular destinations or for secretion. When proteins misfold, selective ER-resident enzymes and chaperones are recruited to rectify the protein-misfolding problem in order to maintain cellular proteostasis. However, when a protein becomes terminally misfolded, it is ejected into the cytosol and degraded by the proteasome via a pathway called ER-associated degradation (ERAD). Strikingly, toxins and viruses can hijack elements of the ERAD pathway to access the host cytosol and cause infection. This review focuses on emerging data illuminating the molecular mechanisms by which these toxic agents co-opt the ER-to-cytosol translocation process to cause disease.
Topics: Animals; Bacterial Infections; Bacterial Physiological Phenomena; Bacterial Toxins; Cytosol; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Host-Pathogen Interactions; Humans; Polyomavirus; Polyomavirus Infections; Protein Transport
PubMed: 26362261
DOI: 10.3109/10409238.2015.1085826 -
Biological & Pharmaceutical Bulletin 2019Proteome profiling based on two-dimensional (2D)-DIGE might be a useful tool for investigating drug-like compounds and the mode of action of drugs. However, obtaining...
Proteome profiling based on two-dimensional (2D)-DIGE might be a useful tool for investigating drug-like compounds and the mode of action of drugs. However, obtaining data for profiling requires high labor costs, and it is difficult to control the reproducibility of spot positions because 2D-DIGE usually requires large-size glass plates and spot alignments are greatly affected by the quality of DryStrips and polyacrylamide gels (PAGs). Therefore, we have developed a novel platform by employing small size DryStrips and PAGs, and an image analysis strategy based on dual correction of spot alignment and volume. Our system can automatically detect a large number of consistent spots through all images. Cytosol fractions of HeLa cells treated with dimethyl sulfoxide (DMSO) or bortezomib were analyzed, 1697 consistent spots were detected, and 775 of them were significantly changed with the treatment. Deviations between different days and lot sets of DryStrips and PAGs were investigated by calculating the correlation coefficients. The mean values of the correlation between days and lot sets were 0.96 and 0.94, respectively. Clustering analysis of all the treatment data clearly separated the DMSO or bortezomib treated groups beyond day deviations. Thus, we have succeeded in developing an easy-to-handle 2D-DIGE system that can be a novel proteome profiling platform.
Topics: Animals; Bortezomib; Cytosol; Dimethyl Sulfoxide; HeLa Cells; Humans; Proteasome Inhibitors; Proteome; Proteomics; Sf9 Cells; Two-Dimensional Difference Gel Electrophoresis
PubMed: 31787721
DOI: 10.1248/bpb.b19-00571 -
Biophysical Journal May 2019The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has...
The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in two-dimensional or three-dimensional single-molecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions.
Topics: Computer Simulation; Cytoplasm; Cytosol; Diffusion; Kinetics; Monte Carlo Method; Single Molecule Imaging; Time Factors
PubMed: 31030884
DOI: 10.1016/j.bpj.2019.03.039 -
Drug Metabolism and Pharmacokinetics 2012Aldehyde oxidase (AO) plays a role in metabolizing many drugs, such as methotrexate and 6-mercaptopurine. We previously showed that AO activity in rat liver rapidly...
Aldehyde oxidase (AO) plays a role in metabolizing many drugs, such as methotrexate and 6-mercaptopurine. We previously showed that AO activity in rat liver rapidly increases from birth, reaching a plateau within 4 weeks, and is regulated at the protein expression level. However, developmental changes of AO activity and protein expression in human liver have not been reported. Here, we investigated the developmental changes and variability of AO in 16 human livers (13 children ranging from 13 days to 12 years old and 3 adults, 17, 34 and 45 years old). Young children (13 days to 4 months after birth) showed little liver AO activity, evaluated in terms of the activities for oxidation of N-1-methylnicotinamide to N-1-methyl-2-pyridone-5-carboxamide and N-1-methyl-4-pyridone-3-carboxamide in liver cytosol. However, these oxidase activities were markedly increased after 4 months, reaching the adult level by about 2 years of age. The AO band density in immunoblotting analysis was well correlated with the AO activity among all subjects (p < 0.01, r(2) = 0.771). Therefore, AO activity in the liver of young children is regulated at the AO protein expression level. Thus, as in rats, the AO activity in humans rapidly increases soon after birth, and is regulated at the protein expression level.
Topics: Adolescent; Adult; Aldehyde Oxidase; Child; Child, Preschool; Cytosol; Female; Humans; Infant; Infant, Newborn; Liver; Male; Middle Aged; Niacinamide; Oxidation-Reduction; Pyridones
PubMed: 22453079
DOI: 10.2133/dmpk.dmpk-11-nt-124 -
ACS Chemical Biology Apr 2023A key limitation for the development of peptides as therapeutics is their lack of cell permeability. Recent work has shown that short, arginine-rich macrocyclic peptides...
A key limitation for the development of peptides as therapeutics is their lack of cell permeability. Recent work has shown that short, arginine-rich macrocyclic peptides containing hydrophobic amino acids are able to penetrate cells and reach the cytosol. Here, we have developed a new strategy for developing cyclic cell penetrating peptides (CPPs) that shifts some of the hydrophobic character to the peptide cyclization linker, allowing us to do a linker screen to find cyclic CPPs with improved cellular uptake. We demonstrate that both hydrophobicity and position of the alkylation points on the linker affect uptake of macrocyclic cell penetrating peptides (CPPs). Our best peptide, , is on par with or better than prototypical CPPs Arg () and under assays measuring total cellular uptake and cytosolic delivery. was also able to carry a peptide previously discovered from an selection, , and a cytotoxic peptide into the cytosol. A bicyclic variant of showed even better cytosolic entry than , highlighting the plasticity of this class of peptides toward modifications. Since our CPPs are cyclized via their side chains (as opposed to head-to-tail cyclization), they are compatible with powerful technologies for peptide ligand discovery including phage display and mRNA display. Access to diverse libraries with inherent cell permeability will afford the ability to find cell permeable hits to many challenging intracellular targets.
Topics: Biological Transport; Cell-Penetrating Peptides; Cytosol
PubMed: 36920103
DOI: 10.1021/acschembio.2c00680 -
STAR Protocols Mar 2021To understand the role of the HIV-1 capsid in viral replication, we developed a protocol to biochemically track capsid in the nucleus during infection. To this end, we...
To understand the role of the HIV-1 capsid in viral replication, we developed a protocol to biochemically track capsid in the nucleus during infection. To this end, we separated HIV-1-infected cells into nuclear and cytosolic fractions. Fractions were analyzed by western blotting for HIV-1 capsid content as well as for nuclear and cytosolic markers to assess the bona fide origin of the fractions. This protocol can be applied in both cycling and non-cycling human cells. For complete details on the use and execution of this protocol, please refer to Selyutina et al. (2020a).
Topics: A549 Cells; Animals; Capsid; Cell Nucleus; Cytosol; Dogs; HEK293 Cells; HIV Infections; HIV-1; HeLa Cells; Humans; Virus Replication
PubMed: 33604582
DOI: 10.1016/j.xpro.2021.100323 -
The Journal of Biological Chemistry Jul 2014Lipid droplets are specific organelles for the storage of triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific...
Lipid droplets are specific organelles for the storage of triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.
Topics: Cytosol; Endoplasmic Reticulum; Enzyme Stability; Intracellular Membranes; Lipase; Protein Structure, Tertiary; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 24847060
DOI: 10.1074/jbc.M114.556944