Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high-content screens, we revisit the question of Tom70 function and considerably expand the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, particularly of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondrion-specifying targeting receptor.
Ray Bradstone, a talented getaway driver, is determined to go straight, be a better parent to his daughter Sally, and make amends with his ex-wife, Lynne. As Ray struggles to find honest work, he agrees to take one last job with his old bank-robbing crew, led by Phillip Larue. Ray changes his mind at the last second, choosing to chaperone a field trip with Sally's class and leaving the thieves without a worthy means of escape. The robbery is a disaster and now Ray must deal with an enraged Larue while driving a school bus full of kids.
The The Chaperone
The chaperone Trigger Factor (TF) from Escherichia coli forms a dimer at cellular concentrations. While the monomer structure of TF is well known, the spatial arrangement of this dimeric chaperone storage form has remained unclear. Here, we determine its structure by a combination of high-resolution NMR spectroscopy and biophysical methods. TF forms a symmetric head-to-tail dimer, where the ribosome binding domain is in contact with the substrate binding domain, while the peptidyl-prolyl isomerase domain contributes only slightly to the dimer affinity. The dimer structure is highly dynamic, with the two ribosome binding domains populating a conformational ensemble in the center. These dynamics result from intermolecular in trans interactions of the TF client-binding site with the ribosome binding domain, which is conformationally frustrated in the absence of the ribosome. The avidity in the dimer structure explains how the dimeric state of TF can be monomerized also by weakly interacting clients.
Where on tau might FKBP12 bind? Using hydrogen, carbon, and nitrogen NMR, co-first author Pijush Chakraborty found that tau remained mostly disordered in the presence of the chaperone protein. However, a spike in signal intensity of two short segments, residues 307 to 311 and 391 to 395, indicated a more stable structure, suggesting FKBP12 might bind there.
Could FKBP12 temper tau aggregation or even neurotoxicity? Indeed, when co-first author Lulu Jiang in the Wolozin lab overexpressed the chaperone in cultured mouse neurons expressing human tau, it reduced phospho-tau and cleaved caspase 3, a neuronal apoptosis marker, induced by tau oligomers. Knocking out FKBP12 did the opposite.
Molecular chaperones are a diverse group of proteins that play critical roles in assisting the folding and assembly of nascent protein chains [1], refolding proteins, assisting protein translocation through membranes [2], and often facilitating protein degradation [3]. Besides physiological processes, these helper proteins also fulfil crucial functions under unfavorable environmental conditions, such as temperature or osmotic stresses, but also in disease conditions and in response to a pathogen attack [3]. Chaperones are involved in the molecular response to stress in plants [4], bacteria [5], and animals [6], and were identified as key players in host-pathogen interaction and in the formation of innate and adaptive immunity [7,8]. During unfavorable environmental conditions, infection or disease, animals rely on a wide variety of chaperones to prevent and reduce the damage caused to cellular compartments and molecules [9]. Overexpression of chaperones in flies and mice so far proved effective in suppressing neurodegeneration [10,11]. In some cardiovascular diseases, cataract, alcoholic hepatitis, cystic fibrosis, phenylketonuria, and in a range of human cancers the protein homeostasis machinery was also shown to be involved as the first line of defense against the disease, thus rendering it a molecular marker for diagnostics [12,13,14]. This makes proteins with chaperone function potential drug targets, due to their function in various pathological and physiological processes inside the cell. Therefore functional and mechanistic characterization is crucial.
The current state of the art in chaperone identification and discovery combines in silico homology screens [14], analysis of transcriptomics data [15], and co-purification with other proteins [16]. Such methods are effective in pooling proteins with stress-related function, but they have limited reproducibility and inherent high error rates of false positives, and require detailed in vitro studies for verification [17]. Methods for chaperone activity testing are under patenting (e.g. application number PCT/US2012/037131), nonetheless, the lack of fast and reliable high-throughput methods for assessing the protective capacity of proteins considerably hinders the discovery and characterization of novel proteins with such functionality. Thus, we propose a semi-high throughput in vitro method for the fast and reliable assessment of chaperone capacity, based on measuring enzyme activity in a 96-well plate format, where the protective activity of a putative chaperone is defined by the retention of enzyme activity during adverse conditions. The proposed method will be of use when a high number of proteins or conditions need to be screened, efficient concentration screens (efficacy) are to be done, or extensive mutation analysis needs to be carried out, for example. In order to show the use of such a method (i.e. to provide proof of principle), we have selected four widely used substrates from the chaperone field: citrate synthase [18], luciferase [19], lactate dehydrogenase [20] and alcohol dehydrogenase [21], optimized the enzymatic assays for the plate reader format and set up the chaperone assay within the plate. We then designed a configuration of the measurements that provides adequate statistical power, and enables us to compare the activity of various enzyme-chaperone mixtures.
In addition to testing the technique on well-established chaperones, we demonstrate the use of the proposed method by characterizing the putative chaperone ERD10. The Early Response to Dehydration protein 10 is a Late Embryogenesis Protein (LEA), described as a dehydrin [22]. Dehydrins are plant proteins primarily involved in cellular and systemic responses to drought stress via various, not fully understood, mechanisms [23]. Kovacs et al. recently explored the putative proteostatic activity of ERD10 and provided formal proof that ERD10 can prevent the aggregation of a range of proteins [24]. Thus, we aim to further explore the protective effect of dehydrins, also demonstrating the advantages of the new technique.
All the protocols follow spectrophotometric reading after the start of the enzymatic reaction. The measurements were performed in a Biotek Synergy Mx microplate reader at wavelengths characteristic for the reactions, until a stable plateau was reached. In all cases, the activity of the corresponding enzyme was defined by the slope of the initial, linear phase of the curve. 100% activity was set to be the activity of the enzyme prior to stress and without the addition of chaperones.
We used the CS activity assay described above to study the protective effect of total protein extracts from E.coli against thermal deactivation of the enzyme. Final amount of 0.02 mg of protein extract per reaction was added to the CS assay instead of a purified chaperone. In order to anticipate the increased background activity of cell extracts, we also added the total protein extract to a blank reaction mix (containing substrates and reporter agent, but no CS). The assay was further performed as already described above, before and after subjecting the protein mix to stress at 44C for 40 min. The enzymatic activity of CS was compared between samples with and without the addition of total E.coli protein extract.
Chaperone assays are widely used to assess the putative function of proteins being expressed or overexpressed in the cell under stress conditions, such as high temperature, oxidative stress, high salinity, UV, etc. The chaperone activity of a protein is determined by the protection of a client protein either against aggregation or the loss of activity evoked by stress. Although chaperone assays are widely used, they usually require tedious experiments due to the high standard deviation of each measurement mostly attributed to experimental, and not biological, error. Classical measurements usually involve the withdrawal of an aliquot from an enzyme assay, followed by a subsequent evaluation of specific properties of the sample and comparison of results between assays with and without the chaperone. Such assays are very sensitive to changes in concentration, to the uniformity of stress applied on the sample and also to other experimental parameters such as time passed between mixing and start of photometric detection. Accordingly, to reduce experimental error of such an assay, both the number of technical replicates and the accuracy of the assay has to be increased.
Activity of an enzyme solution is defined by the initial, maximum velocity of a reaction, measured at 10-fold excess of the substrate over the Km. Under such conditions, the measured reaction velocity is above 90% of the Vmax, and remains linear over a longer period of time, due to the large excess of substrate over the product. In practice, the velocity is measured as the slope of the initial linear phase of the reaction (Fig 1). For comparison of different samples, it is important to define the velocity exactly at the same time point after mixing the components, in order to have an accurate assessment of the effect of the chaperone. Nonetheless, due to technical limitations, the first few seconds of the reaction cannot be observed, which is why the slopes of the curves converge at the negative side of the timeline. The point where the fitted slopes cross shows that a linear fit has been done on the linear phase of the reaction curve. The slopes can then be compared. It is also apparent from Fig 1 that the experimental setup is suitable to measure changes in the velocity (see next section). Confidence intervals of technical repeats illustrate the accuracy of the measurements. 2ff7e9595c
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