To probe the mechanism of the Hsp90 chaperone that is required for the maturation of many signaling proteins in eukaryotes, we analyzed the effects of all individual amino acid changes in the ATPase ...domain on yeast growth rate. The sensitivity of a position to mutation was strongly influenced by proximity to the phosphates of ATP, indicating that ATPase-driven conformational changes impose stringent physical constraints on Hsp90. To investigate how these constraints may vary for different clients, we performed biochemical analyses on a panel of Hsp90 mutants spanning the full range of observed fitness effects. We observed distinct effects of nine Hsp90 mutations on activation of v-src and glucocorticoid receptor (GR), indicating that different chaperone mechanisms can be utilized for these clients. These results provide a detailed guide for understanding Hsp90 mechanism and highlight the potential for inhibitors of Hsp90 that target a subset of clients.
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•Functional requirement at each site of N-Hsp90 revealed by systematic mutagenesis•Conformational constraints depend on amino acid proximity to γ-phosphate of ATP•The v-src kinase imposes more stringent constraints on hydrolysis compared to GR•ATPase domain of Hsp90 uses client-specific features to mature different clients
Mishra et al. now examine the mechanism by which Hsp90 helps clients to mature by analyzing the effects of all individual mutations in the ATPase domain on yeast growth rate. The results indicate that both general and client-specific features of this domain contribute to chaperone function.
Gene-environment interactions have long been theorized to influence molecular evolution. However, the environmental dependence of most mutations remains unknown. Using deep mutational scanning, we ...engineered yeast with all 44,604 single codon changes encoding 14,160 amino acid variants in Hsp90 and quantified growth effects under standard conditions and under five stress conditions. To our knowledge, these are the largest determined comprehensive fitness maps of point mutants. The growth of many variants differed between conditions, indicating that environment can have a large impact on Hsp90 evolution. Multiple variants provided growth advantages under individual conditions; however, these variants tended to exhibit growth defects in other environments. The diversity of Hsp90 sequences observed in extant eukaryotes preferentially contains variants that supported robust growth under all tested conditions. Rather than favoring substitutions in individual conditions, the long-term selective pressure on Hsp90 may have been that of fluctuating environments, leading to robustness under a variety of conditions.
Interactions among mutations within a protein have the potential to make molecular evolution contingent and irreversible, but the extent to which epistasis actually shaped historical evolutionary ...trajectories is unclear. To address this question, we experimentally measured how the fitness effects of historical sequence substitutions changed during the billion-year evolutionary history of the heat shock protein 90 (Hsp90) ATPase domain beginning from a deep eukaryotic ancestor to modern Saccharomyces cerevisiae. We found a pervasive influence of epistasis. Of 98 derived amino acid states that evolved along this lineage, about half compromise fitness when introduced into the reconstructed ancestral Hsp90. And the vast majority of ancestral states reduce fitness when introduced into the extant S. cerevisiae Hsp90. Overall, more than 75% of historical substitutions were contingent on permissive substitutions that rendered the derived state nondeleterious, became entrenched by subsequent restrictive substitutions that made the ancestral state deleterious, or both. This epistasis was primarily caused by specific interactions among sites rather than a general effect on the protein’s tolerance to mutation. Our results show that epistasis continually opened and closed windows of mutational opportunity over evolutionary timescales, producing histories and biological states that reflect the transient internal constraints imposed by the protein’s fleeting sequence states.
ClpXP is a protease involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. To date, however, only a handful of ClpXP substrates have been ...identified. Using a tagged and inactive variant of ClpP, substrates of
E. coli ClpXP were trapped in vivo, purified, and identified by mass spectrometry. The more than 50 trapped proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. Analysis of the sequences of the trapped proteins revealed five recurring motifs: two located at the C terminus of proteins, and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations established that sequences from each motif class targeted proteins for degradation by ClpXP. These results represent a description of general rules governing substrate recognition by a AAA+ family ATPase and suggest strategies for regulation of protein degradation.
Serological assays intended for diagnosis, sero-epidemiologic assessment, and measurement of protective antibody titers upon infection or vaccination are essential for managing the SARS-CoV-2 ...pandemic. Serological assays measuring the antibody responses against SARS-CoV-2 antigens are readily available. However, some lack appropriate characteristics to accurately measure SARS-CoV-2 antibodies titers and neutralization. We developed an Enzyme-linked Immunosorbent Assay (ELISA) methods for measuring IgG, IgA, and IgM responses to SARS-CoV-2, Spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins. Performance characteristics of sensitivity and specificity have been defined. ELISA results show positive correlation with microneutralization and Plaque Reduction Neutralization assays with infectious SARS-CoV-2. Our ELISA was used to screen healthcare workers in Louisville, KY during the first wave of the local pandemic in the months of May and July 2020. We found a seropositive rate of approximately 1.4% and 2.3%, respectively. Our analyses demonstrate a broad immune response among individuals and suggest some non-RBD specific S IgG and IgA antibodies neutralize SARS-CoV-2.
Mechanistic Asymmetry in Hsp90 Dimers Flynn, Julia M.; Mishra, Parul; Bolon, Daniel N.A.
Journal of Molecular Biology/Journal of molecular biology,
09/2015, Letnik:
427, Številka:
18
Journal Article
Recenzirano
Odprti dostop
Hsp90 is a molecular chaperone that facilitates the maturation of signaling proteins including many kinases and steroid hormone receptors. Through these client proteins, Hsp90 is a key mediator of ...many physiological processes and has emerged as a promising drug target in cancer. Additionally, Hsp90 can mask or potentiate the impact of mutations in clients with remarkable influence on evolutionary adaptations. The influential roles of Hsp90 in biology and disease have stimulated extensive research into the molecular mechanism of this chaperone. These studies have shown that Hsp90 is a homodimeric protein that requires ATP hydrolysis and a host of accessory proteins termed co-chaperones to facilitate the maturation of clients to their active states. Flexible hinge regions between its three structured domains enable Hsp90 to sample dramatically distinct conformations that are influenced by nucleotide, client, and co-chaperone binding. While it is clear that Hsp90 can exist in symmetrical conformations, recent studies have indicated that this homodimeric chaperone can also assume a variety of asymmetric conformations and complexes that are important for client maturation. The visualization of Hsp90-client complexes at high resolution together with tools to independently manipulate each subunit in the Hsp90 dimer are providing new insights into the asymmetric function of each subunit during client maturation.
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•Hsp90 mediates proteostasis and plays key roles in evolution and cancer.•Hsp90 is a homodimer that undergoes ATPase-driven conformational changes.•Recent studies have probed how each Hsp90 subunit contributes to function.•Structures with clients and co-chaperones are asymmetric.•ATP hydrolysis is only required in one subunit of the Hsp90 dimer.
•Quantification of the experimental fitness effects for thousands of mutations.•Views into the biophysics of protein folding and function.•Mechanisms of adaptation to new environments including drug ...resistance.•Efforts to predict disease-causing mutations in humans.
Biology has, and continues to be, shaped by evolutionary mechanisms. Within the past decade, local fitness landscapes have become experimentally tractable and are providing new perspectives on evolutionary mechanisms. Powered by next-generation sequencing, the impacts of all individual amino acid substitutions on function have been quantified for dozens of proteins. These fitness maps have been utilized to investigate the biophysical underpinnings of existing protein function as well as the appearance and enhancement of new protein functions. This review highlights emerging trends from this rapidly growing area of research, including an expanded understanding of the biophysical mechanisms underlying existing and new protein function, the roles epistasis and adaptation play in shaping evolution, and the prediction of disease-causing alleles in humans.
Background
Prenatal alcohol exposure (PAE) can result in harmful and long‐lasting neurodevelopmental changes. Children with PAE or a fetal alcohol spectrum disorder (FASD) have decreased white matter ...volume and resting‐state spectral power compared to typically developing controls (TDC) and impaired resting‐state static functional connectivity. The impact of PAE on resting‐state dynamic functional network connectivity (dFNC) is unknown.
Methods
Using eyes‐closed and eyes‐open magnetoencephalography (MEG) resting‐state data, global dFNC statistics and meta‐states were examined in 89 children aged 6–16 years (51 TDC, 38 with FASD). Source analyzed MEG data were used as input to group spatial independent component analysis to derive functional networks from which the dFNC was calculated.
Results
During eyes‐closed, relative to TDC, participants with FASD spent a significantly longer time in state 2, typified by anticorrelation (i.e., decreased connectivity) within and between default mode network (DMN) and visual network (VN), and state 4, typified by stronger internetwork correlation. The FASD group exhibited greater dynamic fluidity and dynamic range (i.e., entered more states, changed from one meta‐state to another more often, and traveled greater distances) than TDC. During eyes‐open, TDC spent significantly more time in state 1, typified by positive intra‐ and interdomain connectivity with modest correlation within the frontal network (FN), while participants with FASD spent a larger fraction of time in state 2, typified by anticorrelation within and between DMN and VN and strong correlation within and between FN, attention network, and sensorimotor network.
Conclusions
There are important resting‐state dFNC differences between children with FASD and TDC. Participants with FASD exhibited greater dynamic fluidity and dynamic range and spent more time in states typified by anticorrelation within and between DMN and VN, and more time in a state typified by high internetwork connectivity. Taken together, these network aberrations indicate that prenatal alcohol exposure has a global effect on resting‐state connectivity.
This study demonstrates that prenatal alcohol exposure alters dynamic functional network connectivity in children with fetal alcohol spectrum disorders. In eyes‐closed and eyes‐open resting states, we found group differences suggesting participants with an FASD exhibit increased dynamic fluidity and dynamic range (i.e. enter more states, change from one meta‐state to another more often, travel greater distances) and spent more time in select meta‐states. These network aberrations indicate prenatal alcohol exposure is having a global effect on resting‐state connectivity.
Prenatal alcohol exposure (PAE) can result in long-lasting changes to physical, behavioral, and cognitive functioning in children. PAE might result in decreased white matter integrity, ...corticothalamic tract integrity, and alpha cortical oscillations. Previous investigations of alpha oscillations in PAE/fetal alcohol spectrum disorder (FASD) have focused on average spectral power at specific ages; therefore, little is known about alpha peak frequency (APF) or its developmental trajectory making this research novel. Using resting-state MEG data, APF was determined from parietal/occipital regions in participants with PAE/FASD or typically developing controls (TDC). In total, MEG data from 157 infants, children, and adolescents ranging in age from 6 months to 17 years were used, including 17 individuals with PAE, 61 individuals with an FASD and 84 TDC. In line with our hypothesis, we found that individuals with PAE/FASD had significantly reduced APF relative to TDC. Both age and group were significantly related to APF with differences between TDC and PAE/FASD persisting throughout development. We did not find evidence that sex or socioeconomic status had additional impact on APF. Reduced APF in individuals with an FASD/PAE may represent a long-term deficit and demonstrates the detrimental impact prenatal alcohol exposure can have on neurophysiological processes.
Adaptor proteins help proteases modulate substrate choice, ensuring that appropriate proteins are degraded at the proper time and place. SspB is an adaptor that delivers ssrA-tagged proteins to the ...AAA+ protease ClpXP for degradation. To identify new SspB-regulated substrates, we examined proteins captured by ClpXP(trap) in sspB(+) but not sspB(-) strains. RseA(1-108), a fragment of a transmembrane protein that regulates the extracytoplasmic-stress response, fits this criterion. In response to stress, RseA is cleaved on each side of the membrane and is released as a cytoplasmic fragment that remains bound in an inhibitory complex with the sigma(E) transcription factor. Trapping experiments together with biochemical studies show that ClpXP functions in concert with SspB to efficiently recognize and degrade RseA(1-108), and thereby releases sigma(E). Genetic studies confirm that ClpX and SspB participate in induction of the sigma(E) regulon in vivo, acting at the final step of an activating proteolytic cascade. Surprisingly, the SspB-recognition sequence in RseA(1-108) is unrelated to its binding sequence in the ssrA tag. Thus, these experiments elucidate the final steps in induction of the extracytoplasmic stress response and reveal that SspB delivers a broader spectrum of substrates to ClpXP than has been recognized.
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