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Pharmacoperones are hydrophobic molecule drugs that enter cells and serve as a molecular framework to cause misfolded mutant proteins to fold properly and adopt a stable conformation compatible with proper intracellular trafficking. Pharmacoperones have successfully been used experimentally to rescue function of some misfolded proteins (enzymes, receptors, channels) that lead to disease. Identification of pharmacoperones by high-throughput screens of drug libraries will likely provide new molecules that may be potentially useful to treat diseases caused by protein misfolding.
Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged primary translation product of SP-C isoforms were generated to test the hypothesis that deposition of hSP-C(I73T) within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, wild-type hSP-C (hSP-C(WT)) colocalized with exogenously expressed human ATP binding cassette class A3 (hABCA3), an indicator of normal trafficking to lysosomal-related organelles. In contrast, hSP-C(I73T) was dissociated from hABCA3 but colocalized to the plasma membrane as well as the endosomal network. Cells expressing hSP-C(I73T) exhibited increases in size and number of cytosolic green fluorescent protein/microtubule-associated protein 1 light-chain 3 (LC3) vesicles, some of which colabeled with red fluorescent protein from the gene dsRed/hSP-C(I73T). By transmission electron microscopy, hSP-C(I73T) cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris, which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-C(I73T) cells exhibited increased expression of Atg8/LC3, SQSTM1/p62, and Rab7, consistent with a distal block in autophagic vacuole maturation, confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-C(I73T) cells showed an impaired degradative capacity for an aggregation-prone huntingtin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and parkin expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-C(I73T) induces an acquired block in macroautophagy-dependent proteostasis and mitophagy, which could contribute to the increased vulnerability of the lung epithelia to second-hit injury as seen in ILD.
Copyright © 2015 the American Physiological Society.
Previous studies from our and other groups have demonstrated that the majority of γ-aminobutyric acid (GABA)(A) receptor subunit mutations produce mutant subunits with impaired biogenesis and trafficking. These GABA(A) receptor mutations include missense, nonsense, deletion, or insertion mutations that result in a frameshift with premature translation-termination codons (PTCs) and splice-site mutations. Frameshift or splice-site mutations produce mutant proteins with PTCs, thus generating nonfunctional truncated proteins. All of these mutant GABA(A) receptor subunits are subject to cellular quality control at the messenger RNA (mRNA) or protein level. These quality-control checkpoints shape the cell's response to the presence of the mutant subunits and attempt to reduce the impact of the mutant subunit on GABA(A) receptor expression and function. The check points prevent nonfunctioning or malfunctioning GABA(A) receptor subunits from trafficking to the cell surface or to synapses, and help to ensure that the receptor channels trafficked to the membrane and synapses are indeed functional. However, if and how these quality control or check points impact the posttranslational modifications of functional GABA(A) receptor channels such as receptor phosphorylation and ubiquitination and their involvement in mediating GABAergic inhibitory synaptic strength needs to be investigated in the near future.
Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.