Learn more: Protein Folding and Proteostasis

A cellular viability assay to monitor drug toxicity
Methods Mol. Biol. 2010, view full abstract in PubMed

A central part of the research in protein misfolding and its associated disorders is the development of treatment strategies based on ensuring cellular protein homeostasis. This often includes testing chemical substances or drugs for their ability to counteract protein misfolding processes and to promote correct folding. Such investigations also include assessment of how the tested chemical substances affect cellular viability, that is, their cytotoxic effect. Investigations of cytotoxicity often require testing several different concentrations and drug exposure times using cells in culture. It is therefore attractive to use a viability test that permits the analysis of many samples with little handling time. This protocol describes a simple and fast methodology to analyze viability of lymphoblastoid cells and to test putative cytotoxic effects associated with exposure to a chemical substance, here exemplified by celastrol. The natural substance celastrol has been used for many years in traditional Chinese medicine and has subsequently been shown to induce transcription of genes encoding molecular chaperones (heat shock proteins) that are involved in promoting folding of cellular proteins. The well-described colorimetric tetrazolium salt (MTT) assay, which monitors metabolic activity of cultured cells, was adapted to analyze the viability of cells exposed to celastrol. After having established a suitable cell seeding density, the dose-dependence and time-course of viability reduction of lymphoblastoid cells treated with celastrol were determined. It was found that 4- and 24-h exposure to 0.8 microM celastrol reduced the viability of lymphoblastoid cells, with the most severe effect observed at 24 h with MTT reductions approaching 30% of non-exposed cells. For a series of incubations for 24 h, it was found that concentrations as low as 0.2 microM were sufficient to affect the viability, and celastrol concentrations of 0.5 microM reduced the MTT reduction rate to approximately half the level displayed by cells receiving vehicle alone.

Independent evolution of the core domain and its flanking sequences in small heat shock proteins
FASEB J. 2010, view full abstract in PubMed

Small heat shock proteins (sHsps) are molecular chaperones involved in maintaining protein homeostasis; they have also been implicated in protein folding diseases and in cancer. In this protein family, a conserved core domain, the so-called alpha-crystallin or Hsp20 domain, is flanked by highly variable, nonconserved sequences that are essential for chaperone function. Analysis of 8714 sHsps revealed a broad variation of primary sequences within the superfamily as well as phyla-dependent differences. Significant variations were found in the number of sHsps per genome, their amino acid composition, and the length distribution of the different sequence parts. Reconstruction of the evolutionary tree for the sHsp superfamily shows that the flanking regions fall into several subgroups, indicating that they were remodeled several times in parallel but independent of the evolution of the alpha-crystallin domain. The evolutionary history of sHsps is thus set apart from that of other protein families in that two exon boundary-independent strategies are combined: the evolution of the conserved alpha-crystallin domain and the independent evolution of the N- and C-terminal sequences. This scenario allows for increased variability in specific small parts of the protein and thus promotes functional and structural differentiation of sHsps, which is not reflected in the general evolutionary tree of species.

Oxidative protein folding in the secretory pathway and redox-signaling across compartments and cells
Traffic. 2010, view full abstract in PubMed

The endoplasmic reticulum (ER) is central for many essential cellular activities, such as the folding, assembly and quality control of secretory and membrane proteins, disulfide bond formation, glycosylation, lipid biosynthesis, Ca(2+) storage and signaling. In addition, this multifunctional organelle integrates many adaptive and/or maladaptive signaling cues reporting on metabolism, proteostasis, Ca(2+) signaling and redox homeostasis. We are beginning to understand how these functions and pathways are integrated with one another to regulate homeostasis at the cell, tissue and organism levels. The mechanisms underlying the introduction of the proper set of disulfide bonds into secretory proteins (oxidative folding) are strictly related to redox homeostasis, ER stress sensing and signaling and provide a good example of the integration systems operative in the early secretory compartment.

Small heat shock proteins and protein-misfolding diseases
Curr. Pharm. Biotechnol. 2010, view full abstract in PubMed

Small heat shock proteins (sHsps) are molecular chaperones ubiquitously distributed in numerous species, from bacteria to humans. A conserved C-terminal "alpha-crystallin" domain organized in a beta-sheet sandwich and oligomeric structure are common features of sHsps. sHsps protect cells against many kinds of stresses including heat shock, oxidative and osmotic stress. sHsps recognize unfolded proteins, prevent their irreversible aggregation and facilitate refolding of bound substrates in cooperation with ATP-dependent molecular chaperones (Hsp70/Hsp40). Mammalian sHsps (HSPBs) are multifunctional proteins involved in many cellular processes including those which are not directly related to protein folding and aggregation. HSPBs participate in cell development and cancerogenesis, regulate apoptosis and control cytoskeletal architecture. Recent data revealed that HSPBs also play an important role in membrane stabilization. Mutation in HSPB genes have been identified, which are responsible for the development of cataract, desmin related myopathy and neuropathies. HSPBs are often found as components of protein aggregates associated with protein-misfolding disorders, such as Parkinson's, Alzheimer's, Alexander's and prion diseases. It is supposed that the presence of HSPBs in intra- or extracellular protein deposits is a consequence of the chaperone activity of HSPBs, however more studies are needed to reveal the exact function of HSPBs during the formation (or removal) of disease-related aggregates.