Removal Of Protein Aggregates Research Articles

Insulin-like Growth Factor-I Prevents the Accumulation of Autophagic Vesicles and Cell Death in Purkinje Neurons by Increasing the Rate of Autophagosome-to-lysosome Fusion and Degradation

Continuous macroautophagic activity is critical for the maintenance of neuronal homeostasis; however, unchecked or dysregulated autophagy can lead to cell death. Cultured Purkinje neurons die by an autophagy-associated cell death mechanism when deprived of trophic support. Here, we report that insulin-like growth factor-I (IGF-I) completely blocked the autophagy-associated cell death of Purkinje neurons. To examine the mechanism by which IGF-I influences autophagy, neurons were infected with adeno-RFP-LC3 and subjected to trophic factor withdrawal, and the size and number of autophagosomes were analyzed by live-cell fluorescence imaging. In control neurons, autophagy occurred at a constitutive low level with most autophagosomes measuring less than 0.75 microm. Trophic factor withdrawal increased the number and size of autophagosomes with most autophagosomes ranging between 0.75 and 1.5 microm and some reaching 1.5-2.25 microm. IGF-I added at the time of trophic factor withdrawal prevented the accumulation of the larger autophagosomes; however, it had no effect on the conversion of LC3, an indicator of autophagy induction. Instead, the rate of autophagosome-to-lysosome fusion measured by colocalization of RFP-LC3 and LysoSensor Green was accelerated by IGF-I. Treating the neurons with bafilomycin A(1) in the presence of IGF-I led to the accumulation of autophagosomes even larger than those induced by trophic factor withdrawal alone, indicating that IGF-I regulates autophagic vesicle turnover. Finally, the effect of IGF-I on autophagy was mediated by an Akt/mTOR-de pend ent and an ERK-independent pathway. These data suggest a novel role for IGF-I in protecting Purkinje neurons from autophagy-associated cell death by increasing autophagy efficiency downstream of autophagy induction.

Autophagy in disease: a double-edged sword with therapeutic potential

Autophagy is a catabolic trafficking pathway for bulk destruction and turnover of long-lived proteins and organelles via regulated lysosomal degradation. In eukaryotic cells, autophagy occurs constitutively at low levels to perform housekeeping functions, such as the destruction of dysfunctional organelles. Up-regulation occurs in the presence of external stressors (e.g. starvation, hormonal imbalance and oxidative stress) and internal needs (e.g. removal of protein aggregates), suggesting that the process is an important survival mechanism. However, the occurrence of autophagic structures in dying cells of different organisms has led to the hypothesis that autophagy may also have a causative role in stress-induced cell death. The identification within the last decade of a full set of genes essential for autophagy in yeast, the discovery of human orthologues and the definition of signalling pathways regulating autophagy have accelerated our molecular understanding and interest in this fundamental process. A growing body of evidence indicates that autophagy is associated with heart disease, cancer and a number of neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's diseases. Furthermore, it has been demonstrated that autophagy plays a role in embryogenesis, aging and immunity. Recently, it has been shown that autophagy can be intensified by specific drugs. The pharmacological modulation of the autophagic pathway represents a major challenge for clinicians to treat human disease.

Autophagy in Ischemic Heart Disease

Autophagy is a major catabolic pathway by which mammalian cells degrade and recycle macromolecules and organelles. It plays a critical role in removing protein aggregates, as well as damaged or excess organelles, to maintain intracellular homeostasis and to keep the cell healthy. In the heart, autophagy occurs at low levels under normal conditions, and defects in this process cause cardiac dysfunction and heart failure. However, this pathway is rapidly upregulated under environmental stress conditions, including ATP depletion, reactive oxygen species, and mitochondrial permeability transition pore opening. Although autophagy is enhanced in various pathophysiological conditions, such as during ischemia and reperfusion, the functional role of increased autophagy is not clear and is currently under intense investigation. In this review, we discuss the evidence for autophagy in the heart in response to ischemia and reperfusion, identify factors that regulate autophagy, and analyze the potential roles autophagy might play in cardiac cells.

CCL2 Protects Prostate Cancer PC3 Cells from Autophagic Death via Phosphatidylinositol 3-Kinase/AKT-dependent Survivin Up-regulation

Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 microm) or the Akt-specific inhibitor-X (Akti-X; 2.5 microm). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr389). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.

Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease

Eukaryotic cells use autophagy and the ubiquitin-proteasome system (UPS) as their major protein degradation pathways. Whereas the UPS is required for the rapid degradation of proteins when fast adaptation is needed, autophagy pathways selectively remove protein aggregates and damaged or excess organelles. However, little is known about the targets and mechanisms that provide specificity to this process. Here we show that mature ribosomes are rapidly degraded by autophagy upon nutrient starvation in Saccharomyces cerevisiae. Surprisingly, this degradation not only occurs by a non-selective mechanism, but also involves a novel type of selective autophagy, which we term 'ribophagy'. A genetic screen revealed that selective degradation of ribosomes requires catalytic activity of the Ubp3p/Bre5p ubiquitin protease. Although ubp3Delta and bre5Delta cells strongly accumulate 60S ribosomal particles upon starvation, they are proficient in starvation sensing and in general trafficking and autophagy pathways. Moreover, ubiquitination of several ribosomal subunits and/or ribosome-associated proteins was specifically enriched in ubp3Delta cells, suggesting that the regulation of ribophagy by ubiquitination may be direct. Interestingly, ubp3Delta cells are sensitive to rapamycin and nutrient starvation, implying that selective degradation of ribosomes is functionally important in vivo. Taken together, our results suggest a link between ubiquitination and the regulated degradation of mature ribosomes by autophagy.

Hepatitis C Virus NS3 Helicase Forms Oligomeric Structures That Exhibit Optimal DNA Unwinding Activity in Vitro

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.

Cell Migration and Signaling Specificity Is Determined by the Phosphatidylserine Recognition Motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.

Patents

Microarray device Cluster ion-exchange membrane Membrane filter housing Hollow-fibre membrane module production Composite membranes Apparatus for producing high-pressure hydrogen Polymeric ultrafiltration or microfiltration membranes Convoluted surface, hollow-fibre membranes Polymer electrolyte membranes Membrane filter for gas treatment Unsupported multi-layer microporous membranes Microporous noble metal material Removing protein aggregates and virus from a protein solution Chemical surface modification of PTFE materials Catalyst-coated membranes

Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography

A new dual-gradient ion exchange chromatographic method was developed to improve the refolding yield of human lysozyme produced inEscherichia coli as an inclusion body. The dissolved and stretched polypeptide chain in a concentrated non-ionic denaturant was adsorbed onto an ion exchange column and induced to refold by gradually decreasing the denaturant concentration and increasing pH in the flowing buffer. The dual gradients of denaturant concentration and pH provided a gradual change of the solution environment along the chromatographic column for the protein to refold, resulting in enhanced activity yield and purity. A post-separation was also studied using size-exclusion chromatography to remove protein aggregates and mis-folded proteins after the refolding step.

The effect of co-overproduction of DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat-aggregated proteins from Escherichia coli ΔclpB mutant cells – new insight into the role of Hsp70 in a functional cooperation with Hsp100

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (σ32-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37°C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and ΔclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the ΔclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.

The effect of co-overproduction of DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat-aggregated proteins from Escherichia coli DeltaclpB mutant cells--new insight into the role of Hsp70 in a functional cooperation with Hsp100.

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma(32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degrees C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and DeltaclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.

Effect of protein aggregates on the complex coacervation between β-lactoglobulin and acacia gum at pH 4.2

The influence of protein aggregates on complex coacervation between β-lactoglobulin (β-lg) and acacia gum (AG) has been studied at pH 4.2 using phase diagrams, electrophoretic mobility measurements, laser light scattering and phase contrast optical microscopy. Removal of protein aggregates by centrifugation (pH 4.75, 1 h, 10,000 g) led to an aggregate-free β-lg dispersion (AF-BLG) exhibiting a narrow polydispersity in molecular masses ( M w∼18,500 and 64,500) and hydrodynamic radii (R h ∼2.51 nm) as determined by GPC and QELS measurements, respectively. Phase diagram determination for the AF-BLG system revealed a two-phase region located in the water-reach corner. In addition, the highest total biopolymer concentration leading to phase separation (4.5 wt%) and the tie-lines shape typically accounted for complex coacervation. The position and extend of the two-phase region differed from that obtained using BLG dispersions (containing protein aggregates). This was partly due to the lowest M w and R h polydispersity of the AF-BLG, but also to the decrease of the electrophoretic mobility ( μ E) of the AF-BLG induced by the protein aggregates elimination (loss of cations in the dispersion). The variation of the μ E of the AF-BLG/AG complexes with pH was similar to that of the BLG/AG ones, but of lower intensity due to the decrease of cations in the system. The size distribution of the BLG/AG complexes obtained at pH 4.2 was mainly governed by the presence of large-sized aggregates (10–50 μm). By contrast, AF-BLG/AG coacervates exhibited a volume average diameter ( d 43) that reached 220 μm for the 2:1 Pr:Ps ratio at pH 4.2, revealing less stability of particles in the system. These results were in agreement with phase contrast optical microscopy that revealed marked differences in the structure of BLG/AG complexes and microparticles as compared to that of AF-BLG/AG coacervates.

The Structure of the β-Propeller Domain and C-terminal Region of the Integrin αM Subunit: DEPENDENCE ON β SUBUNIT ASSOCIATION AND PREDICTION OF DOMAINS

The Structure of the β-Propeller Domain and C-terminal Region of the Integrin αM Subunit: DEPENDENCE ON β SUBUNIT ASSOCIATION AND PREDICTION OF DOMAINS

Immunoaffinity purification and quantification of antibody-toxin conjugates.

Cytotoxic antibody-toxin conjugates made using antibodies and ribs some-inactivating proteins (RIPs) are prepared using chemical crosslinking methods (1,2 and this vol., Chapter 31 ). Gel permeation chromatography is used as a first step to purify conjugate molecules from the reaction mixture. This procedure removes protein aggregates, the excess of RIP employed in the conjugation reaction, and low molecular weight byproducts. However, a significant fraction of the resulting conjugate preparation consists of unconjugated antibody that cannot be completely separated from the conjugate on the basis of size discrimination alone (see chapter 31 ).

Surface Immunoglobulin Receptors of Rabbit Lymphoid Cells

Rabbit lymphoid cells were stained with fluorescein-labelled antiallotype antibodies. The double layer technique was found to be more sensitive than the direct staining. Rabbit B cells are stained only via their surface immunoglobulin (sIg) receptors and not via the receptors for the Fc portion of the IgG. Peritoneal exudate macrophages do not carry sIg receptors and are stained via their Fc receptors. Removal of protein aggregates from the system and use of reagents prepared from F(ab')2 immunoglobulin fragments prevent staining of the macrophages through their Fc receptors. There was a good agreement between the percent of RABELA-positive and sIg-containing spleen cells. In appendix there were approximately 20% more RABELA-positive than sIg-positive cells.

Specific in vitro binding of radiolabeled antibodies to cell surface antigens: Interaction of antihuman thymocyte globulin with human target cells

We have developed a highly sensitive and reproducible in vitro method to measure the rate and amount of binding of radiolabeled antibodies to target cells. We reduced non-specific binding of the antibodies to the target cells approx. 92% by removing protein aggregates from the antibody solutions. Using a specially designed cup that simultaneously allows washing and collection of target cells, cell loss was eliminated. This method was used to study the in vitro binding properties of the IgG fraction of anti-human thymocyte globulin (ATG) to nucleated human cells and to cells of other species. We found the initial rapid uptake of labeled ATG-IgG slowed with prolonged incubation. Incubation temperature and ATG concentration increased the rate of uptake. Sequential absorption studies indicated that intial uptake was due to rapidly binding antibodies. After these antibodies were removed, the rate of binding for antibodies that remained was several fold less than that of the antibodies removed by the initial absorption. Since temperature and the concentration of antibody and target cells can be rigidly controlled, this in vitro model system is ideally suited to quantify optimal conditions and kinetics of antibody binding to cell membrane antigens. Furthermore, the binding properties of antibody subpopulations in an antiserum may be determined by this technique. The maximum antibody binding capacity of various cell types can also be measured using the technique with a precision of ± 14% on replicate determinations.