Free radicals originate from both exogenous environmental sources and as by-products of the respiratory chain and cellular oxygen metabolism. Sustained accumulation of free radicals, beyond a physiological level, induces oxidative stress that is harmful for the cellular homeodynamics as it promotes the oxidative damage and stochastic modification of all cellular biomolecules including proteins. In relation to proteome stability and maintenance, the increased concentration of oxidants disrupts the functionality of cellular protein machines resulting eventually in proteotoxic stress and the deregulation of the proteostasis (homeostasis of the proteome) network (PN). PN curates the proteome in the various cellular compartments and the extracellular milieu by modulating protein synthesis and protein machines assembly, protein recycling and stress responses, as well as refolding or degradation of damaged proteins. Molecular chaperones are key players of the PN since they facilitate folding of nascent polypeptides, as well as holding, folding, and/or degradation of unfolded, misfolded, or non-native proteins. Therefore, the expression and the activity of the molecular chaperones are tightly regulated at both the transcriptional and post-translational level at organismal states of increased oxidative and, consequently, proteotoxic stress, including ageing and various age-related diseases (e.g. degenerative diseases and cancer). In the current review we present a synopsis of the various classes of intra- and extracellular chaperones, the effects of oxidants on cellular homeodynamics and diseases and the redox regulation of chaperones.

Amyloid deposits to the islets of Langerhans are responsible for the gradual loss of pancreatic β-cells leading to type II diabetes mellitus. Human mature islet amyloid polypeptide (hIAPP), a 37-residue pancreatic hormone, has been identified as the primary component of amyloid fibrils forming these deposits. Several individual segments along the entire sequence length of hIAPP have been nominated as regions with increased amyloidogenic potential, such as regions 8-20, 20-29, and 30-37. A smaller fragment of the 8-20 region, spanning residues 8-16 of hIAPP has been associated with the formation of early transient α-helical dimers that promote fibrillogenesis and also as a core part of hIAPP amyloid fibrils. Utilizing our aggregation propensity prediction tools AmylPred and AmylPred2, we have identified the high aggregation propensity of the 8-16 segment of hIAPP. A peptide analog corresponding to this segment was chemically synthesized and its amyloidogenic properties were validated using electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, and polarized microscopy. Additionally, two peptides introducing point mutations L12R and L12P, respectively, to the 8-16 segment, were chemically synthesized. Both mutations disrupt the α-helical properties of the 8-16 region and lower its amyloidogenic potential, which was confirmed experimentally. Finally, cytotoxicity assays indicate that the 8-16 segment of hIAPP shows enhanced cytotoxicity, which is relieved by the L12R mutation but not by the L12P mutation. Our results indicate that the chameleon properties and the high aggregation propensity of the 8-16 region may significantly contribute to the formation of amyloid fibrils and the overall cytotoxic effect of hIAPP.

The maintenance of biomolecules functionality is essential for the assurance of cellular homeostasis. At the proteome level this is achieved by the action of a modular, yet integrated subcellular compartment-specific system which ensures proteome quality control and it is called the proteostasis network (PN). PN is orchestrated by a plethora of different mechanisms and complex protein machines aiming to respond to stressors, recognize and either rescue (via the action of chaperones) or degrade unfolded, misfolded or damaged polypeptides at the two main cellular proteolytic systems, namely the proteasome and the lysosome. We have found that aging is accompanied by increased proteotoxic stress and a reduction of PN modules functionality. Interestingly, proteotoxic stress is also an emerging hallmark of age-related diseases, including tumorigenesis, and we recently proposed that age-related disruption of proteostasis contributes to tumor formation by (among others) increasing genomic instability due to reduced fidelity in processes like DNA replication or repair. We also hypothesized that the carcinogenesis-related increasing proteome instability eventually triggers the reactivation of the PN modules at advanced and/or metastatic stages in order for the tumor to survive and enable its various malignant phenotypes. We are testing these hypotheses by examining the differential regulation of PN components in precancerous and cancerous lesions of tumor biopsies, as well as, in cellular models of step-wise carcinogenesis. Specifically, precancerous and cancerous lesions of the larynx along with normal epithelium were examined by immunohistochemical staining for the expression levels of Apolipoprotein J/Clusterin (CLU); a molecular chaperone that has been implicated in tumor formation, metastasis and tumor cells resistance to chemotherapeutic drugs. Additionally, human bronchial epithelial cells into which various oncogenes were sequentially introduced, as well as, a mouse skin carcinogenesis model have been used to: a) investigate the status of PN components as a response to oncogenic hits and, b) to test the differential sensitivity of tumor vs. normal cells to stressors and to PN modules inhibitors. We found that CLU expression is induced by de novo synthesis in larynx dysplasia. In support, at both human and mouse cellular models of carcinogenesis we noted higher levels and activities of PN modules in advanced tumorigenesis; these phenotypes were accompanied with increased oxidative damage and proteome instability. Interestingly, advanced and/or metastatic mouse tumor cells were more sensitive to disruption of proteostasis (e.g. proteasome inhibition) or increased oxidative load compared to normal cells, while combined proteasome and lysosomal inhibition further sensitized metastatic tumor cells. These observations indicate that while tumor evolves over a cellular landscape of increased proteome instability, tumor cells eventually become “addicted” to higher activities of the PN modules in order to survive. Thus, inhibition of PN components provides a strategy for the development of novel tumor specific therapies. We are currently investigating this option by screening for natural compounds functioning as potent inhibitors of PN modules. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B73. Citation Format: Eirini-Stavroula Komseli, Fabiola Sesti, Konstantinos Evangelou, Christina Cheimonidou, Athanassios Kotsinas, Vassilis Gorgoulis, Ioannis P. Trougakos. Proteostasis network modules as molecular targets for cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B73.