SummarySeveral metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.
Background: Hsp110s are considered as mere nucleotide exchange factors of the Hsp70s. Results: Human cytosolic Hsp110s can use ATP to unfold misfolded polypeptides and act as equal partner with Hsp70 to solubilize stable protein aggregates. Conclusion: Hsp110s are Hsp70-like polypeptide unfolding chaperones. Significance: Hsp110s are powerful disaggregating chaperones that can collaborate with Hsp70s to detoxify misfolding proteins in degenerative diseases.
Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As oversticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins.
Cadmium is a highly poisonous metal and is classified as a human carcinogen. While its toxicity is undisputed, the underlying in vivo molecular mechanisms are not fully understood. Here, we demonstrate that cadmium induces aggregation of cytosolic proteins in living Saccharomyces cerevisiae cells. Cadmium primarily targets proteins in the process of synthesis or folding, probably by interacting with exposed thiol groups in not-yet-folded proteins. On the basis of in vitro and in vivo data, we show that cadmium-aggregated proteins form seeds that increase the misfolding of other proteins. Cells that cannot efficiently protect the proteome from cadmium-induced aggregation or clear the cytosol of protein aggregates are sensitized to cadmium. Thus, protein aggregation may contribute to cadmium toxicity. This is the first report on how cadmium causes misfolding and aggregation of cytosolic proteins in vivo. The proposed mechanism might explain not only the molecular basis of the toxic effects of cadmium but also the suggested role of this poisonous metal in the pathogenesis of certain protein-folding disorders.
a b s t r a c tStress-denatured or de novo synthesized and translocated unfolded polypeptides can spontaneously reach their native state without assistance of other proteins. Yet, the pathway to native folding is complex, stress-sensitive and prone to errors. Toxic misfolded and aggregated conformers may accumulate in cells and lead to degenerative diseases. Members of the canonical conserved families of molecular chaperones, Hsp100s, Hsp70/110/40s, Hsp60/CCTs, the small Hsps and probably also Hsp90s, can recognize and bind with high affinity, abnormally exposed hydrophobic surfaces on misfolded and aggregated polypeptides. Binding to Hsp100, Hsp70, Hsp110, Hsp40, Hsp60, CCTs and Trigger factor may cause partial unfolding of the misfolded polypeptide substrates, and ATP hydrolysis can induce further unfolding and release from the chaperone, leading to spontaneous refolding into native proteins with low-affinity for the chaperones. Hence, specific chaperones act as catalytic polypeptide unfolding isomerases, rerouting cytotoxic misfolded and aggregated polypeptides back onto their physiological native refolding pathway, thus averting the onset of protein conformational diseases.
Bioengineering an in vitro liver model recapitulating the native liver microarchitecture consisting of parenchymal and non-parenchymal cells is crucial in achieving cellular crosstalk and hepatic metabolic functions for accurate hepatotoxicity prediction. Bioprinting holds the promise of engineering constructs with precise control over the spatial distribution of multiple cells. Two distinct tissue-specific liver extracellular matrix (ECM)-based bioinks with excellent printability and rheological attributes are formulated for supporting parenchymal and non-parenchymal cells. A physiologically relevant human vascularized liver model is bioprinted with a novel liver ECM-based bioink laden with human adipose mesenchymal stem cell-derived hepatocyte-like cells (HLCs), human umbilical vein endothelial cells (HUVECs), and human hepatic stellate cells (HHSCs) using an extrusion-based bioprinting approach and validated for hepatotoxicity assessment. The HLC/HUVEC/HHSCladen liver model resembles native alternate cords of hepatocytes with a functional sinusoidal lumen-like network in both horizontal and vertical directions, demonstrating enhanced albumin production, urea synthesis, and cytochrome P450 (CPR) activity. Furthermore, the liver model is evaluated for drug toxicity assessment following 24 h exposure to different concentrations of (i) non-hepatotoxicants aspirin and dexamethasone, (ii) idiosyncratic hepatotoxicant trovafloxacin mesylate, and (iii) clinical hepatotoxicant acetaminophen and troglitazone. A followup cell viability and metabolic competence evaluation by estimating DNA concentration, lactate dehydrogenase activity, and CPR activity revealed a dose-dependent clinically relevant hepatotoxic response. These results corroborated that the developed clinically relevant vascularized liver model is affordable and would aid pharmaceutical companies in speeding up the drug development and provide a robust platform for hepatotoxicity screening.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.