Proteins disaggregation

B., Novel insights into the mechanism of chaperone-assisted protein disaggregation, Biol. Chem. 386, 739-744, 2005. 

Parsed DA, Kowal AS, Singer MA, Lindquist S (1994) Protein disaggregation mediated by heat-shock protein Hspl04. Nature 372 475-478... 

Tessarz P, Mogk A, Bukau B (2008) Substrate threading through the central pore of the Hspl04 chaperone as a common mechanism for protein disaggregation and prion propagation. Mol Microbiol 68 87-97... 

Weibezahn J, Bukau B, Mogk A (2004) Unscrambling an egg protein disaggregation by AAA + proteins. Microb Cell Fact 3 1-12... 

Haslberger T, Zdanowicz A, Brand I, Kirstein J, Turgay K, Mogk A, Bukau B (2008) Protein disaggregation by the AAA + chaperone ClpB involves partial threading of looped polypeptide segments. Nat Struct Mol Biol 15 641-650... 

Cashikar AG, Duennwald M, Lindquist SL (2005) A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hspl04. J Biol Chem 280 23869-23875... 

Photocrosslinkers have been incorporated in E. coli to confirm close contact between specific residues of a protein and its substrate. ClpB, a heat shock protein that aids in the disaggregation and refolding of proteins during the heat shock response, has a conserved aromatic residue (Tyr251) in the central pore of its AAA+ domain, considered to be the main substrate recognition residue. " After Tyr251 in ClpB was replaced with />BpA, biotinylated substrate peptides were shown to be crosslinked upon UV light exposure, but not if BpA was incorporated elsewhere in the AAA+ domain of this protein. 

The relative importance of hepatic microsomal lipid peroxidation versus covalent binding of carbon tetrachloride-derived radicals has been the subject of considerable debate. Since cytochrome P-450 loss has been shown to be related to lipid peroxidation and to covalent binding, each in the absence of the other, both of these early consequences of carbon tetrachloride metabolism may contribute to P-450 destruction. Nevertheless, it is still not clear how these initial events are related to subsequent triglyceride accumulation, polyribosomal disaggregation, depression of protein synthesis, cell membrane breakdown and eventual death of the hepatocytes. Carbon tetrachloride... 

Proteins of egg white denature more rapidly than those of whey protein concentrate (13, 34). However, isolated p-lactoglobulin from the whey concentrate was more susceptible to surface denaturation than egg white ovalbumin. These data suggest that whey contains substances that protect the proteins from surface denaturation and may account for the lower stability of whey protein concentrate foams than those of egg white protein. A balance between the disaggregation effect of select pH values and the tendency toward greater aggregation of proteins at higher heating temperatures were correlated closely with maximum foam stability (13, 15). 

Figure 4. ORD of mitochondrial structural protein in the disaggregated state at pH 11 and the aggregated state at pH 9. Spectra were taken in distilled water, and pH was adjusted with HCl or KOH...

The most obvious fluorescent compound in milk is riboflavin, which absorbs strongly at 440-500 nm and emits fluorescent radiation with a maximum at 530 nm. Riboflavin in whey is measured easily by fluorescence (Amer. Assoc. Vitamin Chemists 1951). Proteins also fluoresce because of their content of aromatic amino acids. Part of the ultraviolet radiation absorbed at 280 nm is emitted at longer wavelengths as fluorescent radiation. A prominent maximum near 340 nm is attributable to tryptophan residues in the protein. Use of fluorescence for quantitation of milk proteins was proposed by Konev and Kozunin (1961), and the technique has been modified and evaluated by several groups (Bakalor 1965 Fox et al. 1963 Koops and Wijnand 1961 Porter 1965). It seems to be somewhat less accurate than desired because of difficulties in disaggregating the caseinate particles and in standardizing instruments. It also involves a basic uncertainty due to natural variations in the proportions of individual proteins which differ in tryptophan content. 

Heating milk to > 100°C for 15 to 20 min, as in the forewarming of milk for evaporated milk manufacture, effectively denatures and complexes whey proteins with casein micelles and causes simultaneous aggregation and disaggregation of these complexed milk protein aggregates (Morr 1975). 

In general, a crude or partially purified extract is electrophoresed on a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) then the protein band is lightly stained and cut out. In the simplest method, the acrylamide gel band is reduced to a pulp, mixed with Freund s adjuvant, and injected. Unfortunately, this technique is not always successful. Its failure can probably be attributed to factors such as the difficulty of disaggregating the acrylamide, the difficulty with which the protein diffuses from the gel, the presence of SDS in large quantities resulting in extensive tissue and cell damage, and finally, the toxicity of the acrylamide... 

Once inside the coil, virulent viruses turn off cellular macromolccular synthesis and disaggregate cellular polyribosomes, thus favoring a shift to viral synthesis. These viruses cause the ultimate destruction of the infected cell. In contrast, moderate viruses may stimulate host DNA, ntRNA, and protein synthesis—a phenomenon which may be of considerable importance in viral carcinogenesis. 

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