Cellular Stress and Protein Misfolding During Aging
The direct evidence that underlying coding polymorphisms have a potential to significantly contribute to conformational disease was recently obtained in C. elegans: expression of either extended polyQ or mutant SOD1 proteins in muscle or neuronal cells of C. elegans lead to the exposure of the ts phenotype at permissive conditions, mediated by the misfolding and loss-of function of ts mutant protein present in the same cell (, ). Furthermore, the misfolding of ts proteins further increased aggregation of the polyQ proteins, thus amplifying the disruption of proteostasis. This effect was most likely caused by the depletion, by the polyQ or mutant SOD1 proteins, of components of chaperone networks that are necessary for maintaining metastable proteins in their folded and functional conformations (B) (; ). A recent finding that many of the modifiers of toxicity of polyQ-expanded ataxin-3 in Drosophila also rescue the generic toxicity of protein misfolding caused by the reduced function of HSP70 () strongly supports the disruption of proteostasis as a mechanism of toxicity. An insight into a potential mechanism by which aggregation-prone proteins may affect the chaperone availability was provided by demonstration that α-synuclein oligomers in vitro inhibited the refolding activity of the HSP70/40 chaperone machinery toward heat- or cold-denatured substrate proteins (). This depletion of chaperone activity was caused by transient weak interactions of α-Synuclein oligomers specifically with HSP40 cochaperones, without their recruitment into the oligomers.
Cellular Stress and Protein Misfolding During ..
We next investigated if the omission of arabinose impacts the cytosolic folding capacity in MGM100 by overexpressing GFP using an isopropyl β--thiogalactopyranoside-inducible vector. We observed a small but consistent shift in newly synthesized GFP from soluble to insoluble fractions upon inhibition of GroEL/GroES expression in exponential cultures (). Hence, inhibition of chaperonin expression has functional consequences, decreasing the cytosolic protein folding capacity.
While this paper was in revision, a complementary study was published. Consistent with our results, the authors found that aminoglycoside induced protein misfolding (). We propose that chaperonin-mediated buffering of aminoglycoside-induced protein misfolding provides a new mechanism for “phenotypic tolerance,” a term that covers various non-inherited antibiotic-resilient phenotypes (). This is important because phenotypic tolerance is known to promote heritable resistance.
03/09/2017 · ER Stress, Protein Misfolding, ..
The chaperone-mediated growth complementation results were confirmed in a system devoid of GFP. In the presence of aminoglycoside, MG1655 growth was rescued () by GroEL/GroES overexpressed at an estimated ~25-fold of non-stressed wild-type levels (, compare lanes 8 and 11). Induction of the DnaK/DnaJ/GrpE expression plasmid did not rescue growth (). Spectinomycin-treated cells showed no growth complementation by GroEL/GroES or DnaK/DnaJ/GrpE overexpression, consistent with chaperonin protection in aminoglycoside-treated cells resulting from a greater capacity for protein misfolding management and not simply a general growth augmentation effect.
enhanced protein synthesis, accumulation of mutated ..
We next measured MG1655 survival after aminoglycoside exposure using a plating assay (). With GroEL/GroES overexpression, the number of cfu remained virtually constant over 4 h of gentamicin treatment. In contrast, when the groES-groEL coding region was deleted from the expression construct (empty control), MG1655 cfu/ml dropped by 2 orders of magnitude (A). Similarly, MG1655 survival decreased with either of two other control plasmids (). One control plasmid overexpressed GFP and did not promote survival. Hence, only chaperone overexpression provided early protection against the aminoglycoside onslaught ensuring survival. Given the inability of DnaK/DnaJ/GrpE to sustain growth in the presence of aminoglycoside (), we were surprised to find this chaperone system to be efficient in promoting cell survival, trailing GroEL/GroES by a modest 2-fold (B). We speculate that slowed bacterial growth allowed the DnaK/DnaJ/GrpE-overexpressing cells to survive within the investigated time period, whereas GroEL/GroES-overexpressing cells supported growth as well as survival because of reduced protein misfolding.