Latent effects of Hsp90 mutants revealed at reduced expression levels

In natural systems, selection acts on both protein sequence and expression level, but it is unclear how selection integrates over these two dimensions. We recently developed the EMPIRIC approach to systematically determine the fitness effects of all possible point mutants for important regions of essential genes in yeast. Here, we systematically investigated the fitness effects of point mutations in a putative substrate binding loop of yeast Hsp90 (Hsp82) over a broad range of expression strengths. Negative epistasis between reduced expression strength and amino acid substitutions was common, and the endogenous expression strength frequently obscured mutant defects. By analyzing fitness effects at varied expression strengths, we were able to uncover all mutant effects on function. The majority of mutants caused partial functional defects, consistent with this region of Hsp90 contributing to a mutation sensitive and critical process. These results demonstrate that important functional regions of proteins can tolerate mutational defects without experimentally observable impacts on fitness

The adaptive potential of the M-domain of yeast Hsp90 [preprint]

Comparing the distribution of fitness effects (DFE) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in other environments. So far, results regarding the cost of adaptation across environments have been mixed, and there were no sufficiently large data sets to map its variation along the genome. Here, we study the DFEs of ≈2500 amino-acid changing mutations obtained from deep mutational scanning of the 118 amino-acid-long middle domain of the heat-shock protein Hsp90 in five environments and at two expression levels. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-M and M-C interdomains and regulation of ATPase-chaperone activity. Despite the diverse and stressful environments, we find that fitness correlates well across environments, with the exception of one environment, diamide. Consistent with these results, we find very little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client binding interfaces or residues that are involved in ATPase chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information

Observation and modelling of ferromagnetic contact-induced spin relaxation in Hanle spin precession measurements

This is the author accepted manuscript. The final version is available from the American Physical Society via http://dx.doi.org/10.1103/PhysRevB.94.094431In the nonlocal spin valve (NLSV) geometry, four-terminal electrical Hanle effect measurements have the potential to provide a particularly simple determination of the lifetime (τ$_{s}$) and diffusion length (λ$_{N}$) of spins injected into nonmagnetic (N) materials. Recent papers, however, have demonstrated that traditional models typically used to fit such data provide an inaccurate measurement of τ$_{s}$ in ferromagnet (FM)/N metal devices with low interface resistance, particularly when the separation of the source and detector contacts is small. In the transparent limit, this shortcoming is due to the back diffusion and subsequent relaxation of spins within the FM contacts, which is not properly accounted for in standard models of the Hanle effect. Here we have used the separation dependence of the spin accumulation signal in NLSVs with multiple FM/N combinations, and interfaces in the diffusive limit, to determine λ$_{N}$ in traditional spin valve measurements. We then compare these results to Hanle measurements as analyzed using models that either include or exclude spin sinking. We demonstrate that differences between the spin valve and Hanle measurements of λ$_{N}$ can be quantitatively modelled provided that both the FM contact-induced isotropic spin sinking and the full three-dimensional geometry of the devices, which is particularly important at small contact separations, are accounted for. We find, however, that considerable difficulties persist, in particular due to the sensitivity of fitting to the contact interface resistance and the FM contact magnetization rotation, in precisely determining λ$_{N}$ with the Hanle technique alone, particularly at small contact separations.This work was funded by Seagate Technology Inc. and the University of Minnesota (UMN) NSF MRSEC under DMR- 1420013, as well as NSF DMR-1104951 and NSF DMR-1507048. L.O’B. acknowledges a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (project no. 299376).Parts of this work were carried out in the UMN Characterization Facility and Minnesota Nano Center, which receive partial support from the NSF MRSEC and NSF NNIN programs, respectively

Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues

The Adaptive Potential of the Middle Domain of Yeast Hsp90

The distribution of fitness effects (DFEs) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in others. So far, results regarding the cost of adaptation across environments have been mixed, and most studies have sampled random mutations across different genes. Here, we quantify systematically how costs of adaptation vary along a large stretch of protein sequence by studying the distribution of fitness effects of the same approximately 2,300 amino-acid changing mutations obtained from deep mutational scanning of 119 amino acids in the middle domain of the heat shock protein Hsp90 in five environments. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-terminal-Middle and Middle-C-terminal interdomains, and regulation of ATPase-chaperone activity. Interestingly, we find that fitness correlates well across diverse stressful environments, with the exception of one environment, diamide. Consistent with this result, we find little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client-binding interfaces, or residues that are involved in ATPase-chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information

MICU1 regulation of mitochondrial Ca(2+) uptake dictates survival and tissue regeneration.

Mitochondrial Ca(2+) uptake through the recently discovered Mitochondrial Calcium Uniporter (MCU) is controlled by its gatekeeper Mitochondrial Calcium Uptake 1 (MICU1). However, the physiological and pathological role of MICU1 remains unclear. Here we show that MICU1 is vital for adaptation to postnatal life and for tissue repair after injury. MICU1 knockout is perinatally lethal in mice without causing gross anatomical defects. We used liver regeneration after partial hepatectomy as a physiological stress response model. Upon MICU1 loss, early priming is unaffected, but the pro-inflammatory phase does not resolve and liver regeneration fails, with impaired cell cycle entry and extensive necrosis. Ca(2+) overload-induced mitochondrial permeability transition pore (PTP) opening is accelerated in MICU1-deficient hepatocytes. PTP inhibition prevents necrosis and rescues regeneration. Thus, our study identifies an unanticipated dependence of liver regeneration on MICU1 and highlights the importance of regulating MCU under stress conditions when the risk of Ca(2+) overload is elevated

The Combined Effect of Oseltamivir and Favipiravir on Influenza A Virus Evolution

Influenza virus inflicts a heavy death toll annually and resistance to existing antiviral drugs has generated interest in the development of agents with novel mechanisms of action. Favipiravir is an antiviral drug that acts by increasing the genome-wide mutation rate of influenza A virus (IAV). Potential synergistic benefits of combining oseltamivir and favipiravir have been demonstrated in animal models of influenza, but the population-level effects of combining the drugs are unknown. In order to elucidate the underlying evolutionary processes at play, we performed genome-wide sequencing of IAV experimental populations subjected to serial passaging in vitro under a combined protocol of oseltamivir and favipiravir. We describe the interplay between mutation, selection, and genetic drift that ultimately culminates in population extinction. In particular, selective sweeps around oseltamivir resistance mutations reduce genome-wide variation while deleterious mutations hitchhike to fixation given the increased mutational load generated by favipiravir. This latter effect reduces viral fitness and accelerates extinction compared with IAV populations treated with favipiravir alone, but risks spreading both established and newly emerging mutations, including possible drug resistance mutations, if transmission occurs before the viral populations are eradicated

An experimental evaluation of drug-induced mutational meltdown as an antiviral treatment strategy [preprint]

The rapid evolution of drug resistance remains a critical public health concern. The treatment of influenza A virus (IAV) has proven particularly challenging, due to the ability of the virus to develop resistance against current antivirals and vaccines. Here we evaluate a novel antiviral drug therapy, favipiravir, for which the mechanism of action in IAV involves an interaction with the viral RNA- dependent RNA polymerase resulting in an effective increase in the viral mutation rate. We utilize an experimental evolution framework, combined with novel population genetic method development for inference from time-sampled data, in order to evaluate the effectiveness of favipiravir against IAV. Evaluating whole genome polymorphism data across fifteen time points under multiple drug concentrations and in controls, we present the first evidence for the ability of viral populations to effectively adapt to low concentrations of favipiravir. In contrast, under high concentrations, we observe population extinction, indicative of mutational meltdown. We discuss the observed dynamics with respect to the evolutionary forces at play and emphasize the utility of evolutionary theory to inform drug development