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PubMed2003

Inclusion of proteins into polyelectrolyte microparticles by alternative adsorption of polyelectrolytes on protein aggregates.

A new method of protein immobilization into polyelectrolyte microparticles by alternative adsorption of the oppositely charged polyelectrolytes on the aggregates obtained by salting out of protein is proposed. The model protein alpha-chymotrypsin (ChT) was included in the polyelectrolyte microparticles obtained by various number of polyelectrolyte adsorption steps (from 1 to 11). The main parameters of ChT inclusion into microparticles were calculated. Scanning electron and optical microscopy were used for characterization of morphology and determination of particle size which was from 1 to 10 micro m in most cases. It was shown that the size and shape of protein-containing particles and protein aggregates used as a matrix were similar. Change in ChT enzymatic activity during entrapment into polyelectrolyte particles and activity of released protein were studied. The effect of pH on release of incorporated proteins was investigated; it was shown that change in pH and the number of polyelectrolyte adsorption steps allows protein release to be manipulated.

Adsorption↗
PubMed2000

Protein aggregation in Huntington's and Parkinson's disease: implications for therapy.

The accumulation of highly insoluble intracellular protein aggregates in neuronal inclusions is a hallmark of Huntington's disease (HD) and Parkinson's disease (PD) as well as several other late-onset neurodegenerative disorders. The aggregates formed in vitro and in vivo generally have a fibrillar morphology, consist of individual beta-strands and are resistant to proteolytic degradation. Although the causal relationship between aggregate formation and disease remains to be proven, the gradual deposition of mutant protein in neurons is consistent with the late-onset and progressive nature of symptoms. Recently, circumstantial evidence from mouse and Drosophila model systems suggests that abnormal protein folding and aggregation play a key role in the pathogenesis of both HD and PD. Therefore, a detailed understanding of the molecular mechanisms of protein aggregation and its effects on neuronal cell death could open new opportunities for therapy.

Animals↗
PubMed1999

The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells.

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.

Bacterial Proteins↗
PubMed1996

IbpA and IbpB, the new heat-shock proteins, bind to endogenous Escherichia coli proteins aggregated intracellularly by heat shock.

IbpA/B, 16 kDa heat-shock proteins were recently described as recognizing heterologous protein inclusion bodies in Escherichia coli cells; the corresponding genes formed an operon regulated by the rpoH gene product, sigma 32 protein (Burland et al (1993) Genomics 16, 551; Allen et al (1992) J Bacteriol 174, 6938; Chuang et al (1993) Gene 134, 1; Chuang and Blattner (1993) J Bacteriol 175, 5242). We have found that IbpA/Bs also recognize endogenous bacterial proteins aggregated intracellularly by heat shock. IbpA/B proteins were isolated and purified from the aggregates (the S fraction), identified by amino acid microsequencing and used as immunogen for anti-IbpA/B serum preparation. Western blotting with the serum showed that in cells growing at 30 degrees C IbpA/B were located in the bacterial outer membrane and appeared in the S fraction after heat shock. Then the cellular level of the IbpA/B proteins increased about 20-fold as estimated by densitometry of the Western blots. In the E coli rpoH strain the level of IbpA/B was higher than in wild type before the heat shock and rose to still higher levels after it. This result pointed to a regulation of ibpA/B operon by another factor, besides that of sigma 32.

Bacterial Proteins↗
PubMed1983

Carotene-cleavage activity in chick intestinal mucosa cytosol: association with a high-molecular-weight lipid-protein aggregate fraction and partial characterization of the activity.

A fluorescent high-molecular weight lipid-protein aggregate was isolated from the cytosol of chick intestinal mucosa or liver by gel filtration on columns of Sepharose 4B or 6B. This aggregate exhibited carotene-cleavage activity. On incubation of this aggregate, dissociation occurred and low-molecular weight fractions containing Cu and Zn and exhibiting carotene-cleavage activity were found. This fraction appeared on sodium dodecyl sulphate polyacrylamide electrophoresis to have a molecular weight of 7000-11000 and resembled the previously described Cu chelatins in amino acid composition. Carotene cleavage may be effected by a copper-zinc metalloprotein of low-molecular weight, associated in intestinal cytosol with a lipid-protein aggregate.

Animals↗
PubMed2003

Hydroperoxy-10,12-octadecadienoic acid stimulates cytochrome P450 3A protein aggregation by a mechanism that is inhibited by substrate.

We recently demonstrated that microsomes from nicardipine-treated rats will form cytochrome P450 3A (CYP3A) aggregates when incubated at 37 degrees C. CYP3A substrates inhibited the protein aggregation and subsequent degradation, suggesting that this process is important in substrate-mediated stabilization of CYP3A. In this paper, we demonstrate that oxidative stress is a key factor in the formation of CYP3A aggregates in incubated microsomes and in a reconstituted system with purified enzymes. Our data further suggest that the effects of oxidative stress are mediated by lipid hydroperoxides, which are efficiently metabolized by CYP3A. In the presence of substrate, the CYP3A-mediated lipid hydroperoxide metabolism is inhibited along with the associated protein aggregation. Therefore, these studies provide a mechanistic model of why CYP3A has a relatively short half-life and how substrates stabilize CYP3A.

Animals↗
PubMed1998

Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria.

Time-dependent aggregation of a plasmid-encoded beta-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 micron3 h-1, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.

Aphthovirus↗
PubMed2006

Nanoparticle-mediated local and remote manipulation of protein aggregation.

The local heat delivered by metallic nanoparticles selectively attached to their target can be used as a molecular surgery to safely remove toxic and clogging aggregates. We apply this principle to protein aggregates, in particular to the amyloid beta protein (Abeta) involved in Alzheimer's disease (AD), a neurodegenerative disease where unnaturally folded Abeta proteins self-assemble and deposit forming amyloid fibrils and plaques. We show the possibility to remotely redissolve these deposits and to interfere with their growth, using the local heat dissipated by gold nanoparticles (AuNP) selectively attached to the aggregates and irradiated with low gigahertz electromagnetic fields. Simultaneous tagging and manipulation by AuNP of Abeta at different stages of aggregation allow both, noninvasive exploration and dissolution of molecular aggregates.

Amyloid beta-Peptides↗
PubMed2003

Neurodegenerative disorders of protein aggregation.

In recent years, it has become increasingly clear that many neurodegenerative diseases involve aggregation and deposition of misfolded proteins such as amyloid beta, tau, alpha-synuclein and polyglutamine containing proteins. This abnormal deposition of misfolded proteins produce malfunctioning of a distinctive set of neurons. It may also induce oxidative and endoplasmic reticulum stress and proteosomal and mitochondrial dysfunction that ultimately leads to neuronal death. While hereditary forms of disorders are caused by genetic mutations, many sporadic cases are likely to be due to genetic and environmental factors. These disorders are progressive in nature. Therefore, treatment is difficult. However, for some diseases, a growing number of treatment options such as drugs, antioxidants, cell transplantation, surgery, rehabilitation procedures and preimplantation diagnosis is available. It should be noted that many of these treatments produce unacceptable risks or adverse effects and they are of only minimal benefit for patients. In future, an understanding of the causes of protein aggregation and genetic and environmental susceptibility factors of a specific individual (or specific individual determinants) may provide a better opportunity for an effective therapeutic intervention.

Alzheimer Disease↗
PubMed2007

Molecular simulation of protein aggregation.

Computer simulation offers unique possibilities for investigating molecular-level phenomena difficult to probe experimentally. Drawing from a wealth of studies concerning protein folding, computational studies of protein aggregation are emerging. These studies have been successful in capturing aspects of aggregation known from experiment and are being used to refine experimental methods aimed at abating aggregation. Here we review molecular-simulation studies of protein aggregation conducted in our laboratory. Specific attention is devoted to issues with implications for biotechnology.

Binding Sites↗
PubMed2005

Role for the alpha-helix in aberrant protein aggregation.

Is the alpha-helix structure capable of triggering the formation of aberrant protein aggregates? To answer this question, we investigate the in vitro aggregation of tau protein in the presence of the helix-inducing agent TFE. Tau is a natively unfolded protein that binds to microtubules and forms aggregates in Alzheimer's disease. We find that full-length tau has residual alpha-helix structure, which is further enhanced by three mutations involved in genetic neurological disorders. TFE concentrations matching an alpha-helical content of 40% in full-length tau and the triple mutant induce the formation of aggregates that are morphologically and structurally heterogeneous. A simple dilution experiment reveals that heterogeneity results from the competition between alpha-helical fibrillar aggregates and more classical amyloid-like aggregates. The alpha-helical aggregates are more resilient to dilution and have the spectroscopic features of alpha-helical coiled coils. We propose a general mechanism by which intrinsically stable alpha-helices can associate into aggregates with only coarse coiled-coil symmetry. In tau, high intrinsic alpha-helix stability and coarse coiled-coil symmetry could be byproducts of its biological function.

Binding Sites↗
PubMed1982

Association of acylglyceride and retinyl palmitate hydrolase activities with zinc and copper metalloproteins in a high molecular weight lipid-protein aggregate fraction from chick liver cytosol.

A lipid-protein aggregate fraction of molecular weight approx. 1.8 . 10(6) was isolated by gel filtration from chick liver cytosol. This aggregate fraction had a hydrated density range of 1.06-1.13, was 45% lipid, contained zinc and copper and was associated with triolein, phosphatidylcholine and retinyl palmitate hydrolase activity. Hydrolytic activities were stimulated by albumin and cholate, but not by dihydroxy bile acids, and inhibited by serine esterase and sulphydryl inhibitors. Incubation of the aggregate with fatty acid-labelled acylglycerides resulted in protein-binding fatty acid fractions with molecular weights of 150,000, 60,000, 13,000 and approx. 2000. Incubation of the aggregate with [3H]retinyl palmitate yielded retinol-containing fractions with molecular weights of 150,000, 60,000 and 15,000. The latter peak appears, on the basis of amino acid composition, to be similar to the cellular retinol-binding protein. In addition, on incubation of the aggregate fraction, zinc and copper peaks are found with molecular weights of 150,000, 60,000 and 12,000-8000. The latter were further purified to yield a copper-rich metalloprotein similar to 'copper-chelatin' and a zinc-rich metalloprotein, possibly zinc-metallothionein. Both these metalloprotein fractions had acyl hydrolase activity which was depressed in zinc-depleted animals. This may provide a possible explanation for the documented nutritional interactions between zinc and retinol.

Animals↗
PubMed2026

Colorimetric gold nanosensors for monitoring protein aggregation: implications for Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia worldwide. It remains a major public health challenge due to the lack of early diagnostic tools and effective disease-modifying therapies. Molecularly, AD is characterized by extracellular amyloid-β (Aβ) plaques and intracellular Tau tangles, as well as soluble oligomers that are likely the neurotoxic species. However, the transient and heterogeneous nature of these oligomers makes them difficult to detect using conventional biosensing approaches. Nanomaterial-based colorimetric biosensors have emerged as promising platforms for detecting protein aggregates and discovering aggregation inhibitors. Specifically, the localized surface plasmon resonance properties of metallic nanomaterials can enable rapid, label-free, and visually detectable colorimetric sensing of molecular interactions. These features can be leveraged to monitor protein aggregation processes in real time and achieve high-throughput screening of aggregation inhibitors, which may collectively enable early detection and timely intervention of AD progression. This Review Article presents the design and engineering of gold-nanomaterial-based colorimetric biosensors for monitoring protein aggregation and highlights the current challenges and emerging opportunities for applying these nanosensors to combat AD.

Journal Article↗
PubMed2006

Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling.

Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation.

Bacterial Proteins↗
PubMed2005

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel.

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.

Bacterial Proteins↗
PubMed2003

A new heat shock gene, AgsA, which encodes a small chaperone involved in suppressing protein aggregation in Salmonella enterica serovar typhimurium.

We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein) in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. The -10 and -35 regions upstream of the transcriptional start site of the agsA gene are characteristic of sigma(32)- and sigma(72)-dependent promoters. AgsA was strongly induced by high temperatures. The similarity between AgsA and the other two sHsps of Salmonella serovar Typhimurium, IbpA and IbpB, is rather low (around 30% amino acid sequence identity). Phylogenetic analysis suggested that AgsA arose from an ancient gene duplication or amplification at an early evolutionary stage of gram-negative bacteria. Here we show that overproduction of AgsA partially complements the DeltadnaK52 thermosensitive phenotype and reduces the amount of heat-aggregated proteins in both DeltadnaK52 and DeltarpoH mutants of Escherichia coli. These data suggest that AgsA is an effective chaperone capable of preventing aggregation of nonnative proteins and maintaining them in a state competent for refolding in Salmonella serovar Typhimurium at high temperatures.

Amino Acid Sequence↗
PubMed2005

Co-translational protein aggregation after transient cerebral ischemia.

Transient cerebral ischemia leads to irreversible translational inhibition which has been considered as a hallmark of delayed neuronal death after ischemia. This study utilized a rat transient cerebral ischemia model to investigate whether irreversible translational inhibition is due to abnormal aggregation of translational complex, i.e. the ribosomes and their associated nascent polypeptides, initiation factors, translational chaperones and degradation enzymes after ischemia. Translational complex aggregation was studied by electron microscopy, as well as by biochemical analyses. A duration of 15 or 20 min of cerebral ischemia induced severe translational complex aggregation starting from 30 min of reperfusion and lasting until the onset of delayed neuronal death at 48 h of reperfusion. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons. After ischemia, most ribosomes were clumped into large abnormal aggregates in neurons destined to die. Translational complex components consisting of small ribosomal subunit protein 6, large subunit protein 28, eukaryotic initiation factor-3eta, co-translational chaperone heat shock cognate protein 70 and co-chaperone HSP40-Hdj1, as well as co-translational ubiquitin ligase c-terminus of hsp70-interacting protein were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational components were also highly ubiquitinated. To our knowledge, irreversible aggregation of translational components has not been reported after brain ischemia. This study clearly indicates that ischemia damages co-translational chaperone and degradation machinery, resulting in irreversible destruction of protein synthesis machinery by protein aggregation after ischemia.

Animals↗
PubMed1981

Protein involvement in structural transition of erythrocyte ghosts. Use of thermal gel analysis to detect protein aggregation.

In this study, it is shown that systematic temperature-induced protein aggregation occurs on the erythrocyte membrane by intermolecular disulfide bond formation. Specific protein bands disappear from acrylamide gel profiles over rather narrow temperature regions. The aggregation appears to be the result of irreversible structural transitions of the membrane, which can be seen in a sensitive scanning calorimeter. When this method of thermal gel analysis is used, the results suggest that spectrin is a participant in the A transition, that bands 2.1, 4.1, and 4.2 and the cytoplasma portion of 3 are involved in the B transition, and that the transmembrane portion of band 3 may undergo changes in the C transition, previously shown to occur in the anion transport domain of the membrane. The aggregation of specific proteins in the narrow temperature region of these transitions persists as the transitions are moved around on the temperature axis by varying solution conditions. The assignment of particular proteins to specific transitions is reinforced by selective extraction of membrane proteins. Large variations in both the calorimetry and the aggregation pattern occur as salt concentration is increased from 77 mosm to 310 mosm, which is manifested in the splitting of the B transition into two separate transitions, B1 and B2. It is speculated that this occurs as the result of a structural change which may involve components of the cytoskeletal network.

Calorimetry↗