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PubMed1977

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities.

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0 degrees C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function. The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.

Biological Transport, Active↗

PubMed1992

Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.

Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.

Amino Acids↗

PubMedDate not supplied

[Radiation-induced aggregation of proteins: binding of amino acids to myoglobin (author's transl)].

When myoglobin is irradiated in the presence of amino acids, the most radiation-reactive species, like the aromatic and sulfur-containing amino acids, will bind preferentially to the protein. The radiation-induced binding is strongly dependent on the concentration of protein and amino acid. Subsequent to irradiation of myoglobin in the presence of radioactively labelled tryptophan followed by tryptic hydrolysis, only a single radioactive spot was detected on the fingerprint. The binding of amino acids is thus not randomly distributed over the protein molecule but occurs at specific reactive sites.

Amino Acids↗

PubMed1979

Rearrangements in the course of ribonuclease hydrolysis of pre-messenger ribonucleoproteins. A warning.

A study of the action of ribonuclease on 30--50-S monoparticles prepared from pre-messenger ribonucleoprotein (pre-mRNA . protein) was started in order to elucidate the structure of monoparticles. A ribonucleoprotein complex containing mostly 30000--38000-Mr proteins of pI 7--9 (alpha class) persisted under conditions where other proteins (23000--110000 Mr, pI 5--8.5, beta class) were relased. An unexpected increase of sedimentation coefficient accompanied the formation of the ribonucleoprotein complex. The extent of increase varied with the initial size of the monoparticles, reaching 45% for 30-S monoparticles. The ribonucleoprotein complexes designated here as 40--45-S alpha-ribonucleoproteins were more homogeneous in size than the original monoparticles. Electron microscopic examination showed that the sedimentation shift corresponded to an increase of the actual size of the particles, not to flattening or change of shape. Therefore, the 40--45-S alpha-ribonucleoprotein is not a pre-existing unit of pre-mRNA . protein but arises from specific rearrangements probably between small alpha ribonucleoproteins formed by fragmentation of monoparticles. In addition to the 40--45-S alpha-ribonucleoproteins, large protein aggregates corresponding to 15% of the monoparticle proteins were formed upon ribonuclease hydrolysis. Their major proteins were neutral, suggesting that the aggregates might be precipitates of proteins at pH close to the pI. Ribonuclease being a widespread cellular enzyme, partial rearrangements may occur during preparation and handling of pre-mRNA . protein. It is particularly crucial to remark that the 40--45-S alpha-ribonucleoprotein which does not pre-exist might be mistaken for a pre-mRNA . protein unit.

Centrifugation, Density Gradient↗

PubMed2025

A rare genetic variant confers resistance to neurodegeneration across multiple neurological disorders by augmenting selective autophagy.

The study of disease modifiers is a powerful way to identify patho-mechanisms associated with disease. Using the strong genetic traits of Huntington's disease (HD), we identified a rare, single-nucleotide polymorphism (SNP) in WDFY3 associated with a delayed age of onset of up to 23 years. Remarkably, the introduction of the orthologous SNP into mice recapitulates this neuroprotection, significantly delaying neuropathological and behavioral dysfunction in two models of HD. The SNP increases expression of the protein autophagy-linked Fab1, YOTB, Vac1, and EEA1 (FYVE) protein (Alfy), an autophagy adaptor protein for the clearance of aggregated proteins, whose ectopic overexpression is sufficient to capture the neuroprotective effects of the variant. Increasing Alfy expression protects not only against HD but also against the toxicity due to phospho-α-synuclein and AT8-positive accumulation. By combining human and mouse genetics, we have uncovered a pathway that protects against multiple proteinopathies, revealing a much-sought-after, shared therapeutic target across a broad range of neurodegenerative diseases.

Animals↗

PubMed1976

Assembly of tobacco mosaic virus.

The assembly of tobacco mosaic virus requires the presence of a particular protein aggregate, the disk. During the nucleation, a specific region of the RNA interacts with a single disk, to bring about a necessarily cooperative transition from the paired two-layer structure to a short segment of nucleo-protein helix. There is a high selectivity for this region of the TMV RNA, because of the many nucleotides bound at once, and other nucleotide sequences appear only to bind by a different mechanism. Elongation of the nucleated rods can continue with either further disks or the less aggregated 'A-protein' as the protein source, but the continued cooperativity inherent with disks would have some advantages. The rates of the two processes have been separately determined and growth is faster when disks are still present. New experiments show that the breakdown of disks to yield A-protein is relatively slow and it is concluded that virus growth from disks could not proceed through a prior breakdown in solution, but must involve the direct interaction of the disk with the growing nucleoprotein rod. The detailed mechanism of disk addition is not understood but it may involve a directed breakdown, since there is also evidence for the existence of a non-equilibrium form of A-protein which has aggregation kinetics distinct from those of equilibrium A-protein. Some implications for the general assembly pathways of viruses both of the specificity and of the assembly/disassembly cycle during the viral infection are considered.

Kinetics↗

PubMed1992

Ubiquitin is associated with aggregates of arginine modified proteins in injured nerves.

Crush injury to rat sciatic nerves results in a 10-fold increase in the post-translational arginylation of proteins. In other systems, N-terminal arginylation leads to ubiquitination and proteolysis of the arginylated proteins. In the present experiments, proteins obtained from the 150 kg supernatant of crushed sciatic nerves were posttranslationally modified by 3H-arginine. These arginine modified proteins formed aggregates (precipitated at 20 kg) which then partially separated by SDS-PAGE were immunoreactive to a monoclonal antibody to ubiquitin. The results indicate that following injury to sciatic nerves, certain proteins are arginylated and ubiquitinated, probably targeting them for degradation. It is likely that these reactions help to rid cells of proteins damaged by the crush which would otherwise be cytotoxic.

Animals↗

PubMed1977

Griseofulvin-induced aggregation of microtubule protein.

Griseofulvin (7-chloro-2',4,6-trimethoxy-6'-methylspiro[benzofuran-2(3H),1'-[2]cyclohexene]-3,4'-dione) induces aggregation of microtubule protein at 0 degrees C. This aggregate contains approx. 90% of the microtubule-associated proteins originally present in the microtubule protein. The supernatant obtained after removal of the griseofulvin-induced aggregate does not form microtubules on warming at 37 degrees C. Addition of the griseofulvin-aggregated protein to this supernatant and warming to 37 degrees C gives rise to a limited amount of microtubule assembly. The possible involvement of griseofulvin-induced aggregation of microtubule protein at 0 degrees C in the inhibition by griseofulvin of microtubule assembly in vitro is discussed.

Animals↗

PubMed1977

Spontaneous, reversible protein cross-linking in the human erythrocyte membrane. Temperature and pH dependence.

Changes in pH significantly affect the morphology and physical properties of red cell membranes. We have explored the molecular basis for these phenomena by characterizing the pattern of protein disulfide cross-linkages formed spontaneously in ghost exposed to acid pH or elevated temperature (37 degrees C). Protein aggregation was analyzed by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. incubation of ghosts at pH 4.0 to 5.5 (0-4 degrees C) yielded (i) complexes of spectrin and band 3, (ii) complexes of actin and band 3, (iii) band 3 complexes, i.e. dimer and trimer, and (iv) heterogeneous aggregates involving spectrin, band 3, band 4.2, and actin in varying proportions. Aggregation was maximal near the isoelectric points of the major membrane proteins, and appeared to reflect (i) the aggregation of intramembrane particles including band 3 and (ii) more intimate contact between spectrin-actin meshwork and band 3.

Actins↗

PubMed1992

Regulation of E2F/cyclin A-containing complex upon retinoic acid-induced differentiation of teratocarcinoma cells.

Retinoic acid-induced differentiation of mouse P19 teratocarcinoma cells is accompanied by alterations in the level of E2F transcription factor. P19 stem cells contain free, uncomplexed E2F and an E2F complex termed E2F/stem. This stem cell complex is a heterotrimeric protein aggregate consisting of E2F transcription factor, E2F-binding protein (E2F/bp1), and cyclin A. Retinoic acid treatment converts P19 stem cells into differentiated neurons, glial cells, and fibroblasts. The presented experiments clearly show that the level of uncomplexed E2F gradually decreases upon differentiation, and fully differentiated cells do not contain free E2F. In addition, the stem cell-specific E2F aggregate is converted into a smaller complex, termed E2F/diff. This smaller complex, which is specific for differentiated cells, does not contain cyclin A and consists of E2F transcription factor associated with E2F/bp1. Finally, the role of E2F complexes in the cessation of cell proliferation, which accompanies P19 cell differentiation, is discussed.

Animals↗

PubMed2026

Riboregulation: a non-canonical tau function.

Almost since its discovery, tau protein has perplexed scientists and clinicians with its varied roles in physiology as well as its appearance as phosphorylated protein aggregates of various structures in many neurodegenerative diseases. Tau plays a role in microtubule stabilization, but from the earliest of studies, tau has also been observed to bind to RNA, with recent research suggesting tau has a higher affinity for some RNA species compared to microtubules. In the context of disease, tau dysfunction potentiates disruptions to RNA metabolism, including the perturbation of mRNA splicing, impairment of translation, de-repression of transposable elements, and alteration of RNA export and degradation. Tau aggregates directly sequester diverse RNA species and RNA binding proteins. Emerging evidence reinforces the characterization of tau as an RNA binding protein, highlighting questions about both the physiological and disease-related functions of this direct RNA binding. The disparate structure of tau in normal and various disease states makes teasing apart the various impacts on RNA and regulation a more difficult puzzle requiring future study. In this review, we summarize the evidence for tau's role in RNA biology, including as an RNA binding protein.

tau Proteins↗

PubMed1976

Testosterone-binding protein in reproductive tracts of fetal rats.

Testosterone-binding proteins may mediate the induction of Wolffian duct differentiation by testicular testosterone. The presence of such protein(s) was sought in reproductive tracts of 14.5-21.5-day-old fetal rats. Supernatant fractions (127,000 x g) were equilibrated with [3H]T) +/- radioinert testosterone in Tris - HCl:EDTA buffer, pH 7.4, (approximately 0.1 mg protein/0.5 ml) at 4 C for 16 hours. Bound and free (3H)T were separated by charcoaldextran adsorption or Sephadex G-100 gel filtration. The results with 14.5-15.0-day-old tracts were: a) specific binding to protein was saturated with increasing (3H)T concentration; b) Scatchard plot analysis indicated the presence of a single class of binding sites with high affinity (apparent Kd = 2 nM) and limited capacity (approximately 16 fmol/mg protein) for (3H)T; c) specific uptake was limited to (3H)T and (3H)5 alpha-dihydrotestosterone; d) (3H)T uptake by the tract supernatant was tissue-specific; e) pronase treatment abolished binding capacity for (3H)T; f) bound radioactivity consisted solely of (3H)T; and g) the mesonephric and ductal segment of the genital tract specifically binds (3H)T. The data demonstrate binding protein(s), specific for testosterone and possibly dihydrotestosterone, in the genital ducts of 14.5-15-day-old fetal rats. (3H)T binding to genital duct supernatants from male but not from female fetuses increased about 5-fold between 14.5 and 20.5 days of gestation. Upon Sephadex G-100 gel filtration, radioactivity was confined to the macromolecular fraction appearing in the void volume. Nuclear fractions, obtained from intact ducts incubated with (3H)T at 30 C but not at 0 C contained radioactivity. These observations are compatible with the existence of a cytoplasmic testosterone receptor or carrier protein aggregate. We have thus concluded that testosterone-binding proteins are present in the genital ducts of rat fetuses and that, in the male, their concentrations increase with progressive Wolffian duct differentiation.

Animals↗

PubMed1992

Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli.

Newly synthesized proteins aggregate extensively in Escherichia coli rpoH mutants, which are deficient in the heat shock proteins (hsp). Overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation. If expressed together, the four hsp are effective at physiological concentrations. Our data suggest that the GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins.

Bacterial Proteins↗

PubMed1992

Initial characterization of heat-induced excess nuclear proteins in HeLa cells.

Exposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two-dimensional gel electrophoresis. The abundance of cytoskeletal elements which co-purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non-heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat-induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus.

Cytoskeletal Proteins↗

PubMed1976

The identification of myosin in rabbit hepatocytes.

A myosin-like protein was identified in isolated rabbit liver cells. It was extracted with high-ionic-strength buffer containing ATP, and purified by gel filtration in the presence of iodide. The myosin polypeptide was indistinguishable in size from the heavy chain of muscle myosin as determined by electrophoresis on polyacrylamide gels and gel filtration in the presence of sodium dodecyl sulfate. The hepatic myosin had an amino acid composition similar to that of muscle myosin, but lacked 3-methylhistidine. The Mg2+ -ATPase of the myosin was not activated by muscle actin. At low ionic strength, in the presence of Mg2+, the protein aggregated to form bipolar filaments 0.3 mum in length. A protein which resembled muscle actin in size and amino acid composition was extracted along with the myosin. Based on scans of stained sodium dodecyl sulfate polyacrylamide gels, the myosin content was estimated as 0.3% to 0.4% of the cell protein. The actin-like component was present in approximately ten-fold excess by weight. This ratio suggests that the organization and function of myosin in the hepatocyte is very different from that in the muscle cell.

Adenosine Triphosphatases↗

PubMed1978

Over two hundred polypeptides resolved from the human erythrocyte membrane.

A modification of O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis has allowed for the resolution of erythrocyte membranes showing up to 200 individual components. Data is presented which indicates that this protein heterogeneity is not produced by artifactual protein-protein aggregation, ednogenous protease activity of secondary charge modification. Similar patterns are obtained when the samples are added to the unpolymerized isoelectric focusing gel, and isolated and stored in protease inhibitor. Individual spots could be eluted off of stained gels, resolubilized under extreme detergent solubilization conditions and run on one-dimensional gels; these run as sodium dodecyl sulfate in the solubilization procedure. The method chosen for solubilization prior to isoelectric focusing appears to cause selective aggregation of all or most of the spectrin and band 3 proteins. This further allows for excellent resolution of more components.

Electrophoresis, Polyacrylamide Gel↗

PubMed1991

High-level secretion of human apolipoprotein E produced in Escherichia coli: use of a secretion plasmid containing tandemly polymerized ompF-hybrid gene.

A gene encoding the mature form of human apolipoprotein E (h-apoE) was fused to the secretion signal coding sequence of the Escherichia coli major outer membrane protein F (ompF) which was preceded by a consensus Shine-Dalgarno sequence. Two copies of this hybrid gene were inserted tandemly into an expression vector and expressed in E. coli under the transcriptional control of two tac promoters regulated by lac repressors. By the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to the growth media, cells synthesized h-apoE at the level of 27.2 micrograms per A600 and up to 22% of the total cellular protein. The h-apoE produced by E. coli was processed precisely, secreted into the periplasmic space and formed protein aggregates there. However, despite aggregation, they were easily dissolved in water and actively formed protein-lipid complexes with dimyristoyl phosphatidyl choline (DMPC). These results demonstrated that E. coli cells are able to synthesize and secrete a large amount of active h-apoE using a prokaryotic signal sequence.

Amino Acid Sequence↗

PubMed1979

The role of glyoxylate in the regulation of biodegradative threonine dehydratase of Escherichia coli.

The activity of biodegradative threonine dehydratase of Escherichia coli K12 was reversibly inhibited by glyoxylate in the presence of AMP. Kinetic analysis showed that the inhibition was mixed with respect to L-threonine and competitive in terms of AMP; the inhibitory effect of glyoxylate was less pronounced at high protein concentrations. Incubation of dehydratase with L-threonine shifted the absorption maximum of the enzyme-bound pyridoxal phosphate from 413 to 425 nm; addition of glyoxylate completely prevented the threonine-mediated spectral shift. In addition to the inhibitory effect, incubation of purified enzyme with glyoxylate resulted in a progressive, irreversible inactivation of the enzyme and formation of inactive protein aggregates. The rates of inactivation were decreased with increasing concentrations of protein and AMP. During inactivation by glyoxylate, the 413-nm absorption maximum of the native enzyme was replaced by a new peak at 385 nm. Experiments with [14C]glyoxylate showed a rapid binding of 1 mol of glyoxylate per 147,000 g followed by a slow binding of 3 additional mol of glyoxylate; the glyoxylate-protein linkage was stable to acid precipitation and protein denaturants. Competition binding experiments revealed that pyruvate (which also inactivated the E. coli enzyme, Feldman, D.A., and Datta, P. (1975) Biochemistry 14, 1760-1767) did not interfere with the binding of glyoxylate or vice versa, suggesting that the two keto acids may occupy separate sites on the enzyme molecule. Nevertheless, experiments on enzyme inactivation using glyoxylate plus pyruvate reveal mutual interactions between these ligands in terms of lack of additive effect, retardation in the spectral shift due to glyoxylate, and stabilization of the enzyme in the presence and absence of AMP. We conclude from these results that the control of biodegradative threonine dehydratase is governed by a complex set of regulatory events resulting from reversible and irreversible association of these effectors with the enzyme molecule.

Adenosine Monophosphate↗