Intracellular folding

Heat-shock proteins (Hsps) are proteins expressed virtually in all organisms as a response of exposure to a stress, such as elevated temperature (fever), protein degradation, mechanical or chemical stress. As chaperone proteins they are concerned with the intracellular folding and refolding, assembly and translocation of damaged proteins. 

Allen S, Naim HY, Bulleid NJ (1995), Intracellular folding of tissue-type plasminogen activator. Effects of disulfide bond formation on N-linked glycosylation and secretion, J. Biol. Chem. 270 4797-4804. 

The Nina A protein of Drosophila is also related to the cyclophilins. It possesses a hydrophobic extension of about 30 residues at its carboxy terminus, which is probably used to anchor the protein in the ER membrane (Shieh et al, 1989 Schneuwly et al, 1989). The function of the NinaA protein is related to the intracellular folding and/or transport of certain classes of rhodopsin molecules in the Drosophila eye (Stamnes et al, 1991 Colley a/., 1991). 

The intracellular folding mechanism of the P22 tailspike has been extensively characterized by genetic analysis (Figure 3). The maturation process of tailspike protein inside the cell proceeds through several defined intermediate stages. Three newly synthesized polypeptide chains first fold into conformations which are ready for association and then fold/associate to a trimeric intermediate, the protrimer, and finally fold into the native trimer 34,44). The half time for the monomeric chains to fold into a SDS- resistant native trimer in vivo at 30°C is about 5 min (56). 

Figure 3. Intracellular folding pathway of P22 tailspike proteins. The newly synthesized wild type or mutant polypeptide chains at 30°C first fold into partially folded monomeric intermediates. These species fold and associate to form a protrimer intermediate. Further folding results in a thermostable native tailspike. At 40°C, the folding is inhibited and tsf mutants act by blocking an early step in chain folding, prior to association. However, if infected cells are shifted to 30 C, the mutant chains continue through the productive pathway.

Cell Disruption Intracellular protein products are present as either soluble, folded proteins or inclusion bodies. Release of folded proteins must be carefully considered. Active proteins are subject to deactivation and denaturation, and thus require the use of gentle conditions. In addition, due consideration must be given to the suspending medium lysis buffers are often optimized to promote protein stability and protect the protein from proteolysis and deactivation. Inclusion bodies, in contrast, are protected by virtue of the protein agglomeration. More stressful conditions are typically employed for their release, which includes going to higher temperatures if necessaiy. For native proteins, gentler methods and temperature control are required. 

As stated above, calcium is an extremely important cellular ion for several cellular functions. The concentration of calcium in human extracellular fluid is about 2.5 mM, while the intracellular concentration is only 100-200 nM depending on the cell type. Thus, there is 10 000-20 000 fold concentration difference between the cell interior and exterior that has to be maintained by cellular pumping mechanisms. This requires a large amount of energy. " ... 

Chaperones bind to exposed hydrophobic surfaces of polypeptide substrates, and through either ATP-dependent or ATP-independent mechanisms facilitate the folding/assembly, intracellular transport, degradation, and activity of polypeptides. 

Immunophillins are abundant proteins that catalyze the cis-trans isomerization of proline residues within proteins, generally to aid in protein folding. Immunophillins are not essential proteins, are the intracellular binding proteins of several immunosuppressive drugs. Cyclosporin A exerts its action after binding to cyclophilin. Tacrolimus and sirolimus predominantly bind to the protein FKBP-12 (FK binding protein-12). 

Intracellular Ca2+-levels are controlled by release into, and removal from, the cytoplasm (Fig. 1). Ca2+-pumps in the plasma membrane and endoplasmic reticulum (ER the Ca2+-store in a cell) keep cytoplasmic Ca2+-levels low (about 0.1 pmol/L in resting cells) and generate a 10,000-fold concentration gradient across membranes (because extracellular Ca2+ is in the millimolar range). Upon stimulation, Ca2+ enters the cytosol of the cell via Ca2+-channels (plasma membrane) or via Ca2+-channels in the ER, leading to the activation of a great variety of Ca2+-dependent processes in the cell. 

It is important to note that the intracellular levels of NTP are approximately 100-fold higher than the dNTP levels. Therefore, much higher median effective... 

Sub-cellular fractionation of five strains reveal the same numbers of bands. The distribution of PG activity in sub-cellular organelles was broadly similar in these five strains. PG activity was detected in low-density vesicles, vacuoles and ER fractions in samples harvested during the early exponential phase of growth. However, PG levels were always lower (at least 1.5 fold) than those found in wild type. Cells of the mutants harvested during stationary phase of growth showed that 84% of total intracellular PG activity was located in the vesicle fraction. No intracellular PG activity was found in stationary phase wild type cells. 

Intracellular products can be present either as folded, soluble proteins or as dense masses of unfolded protein (inclusion bodies). For these products, it is first necessary to concentrate the cell suspension before effecting release of the product. Filtration can result in a suspension of cells that can be of any desired concentration up to 15 to 17 percent and that can be diafiltered into the desired buffer system. In contrast, the cell slurry that results from centrifugation will be that of... 

---