Intracellular spatially-targeted chemical chaperones increase native state stability of mutant SOD1 barrel.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder with currently no cure. Central to the cellular dysfunction associated with this fatal proteinopathy is the accumulation of unfolded/misfolded superoxide dismutase 1 (SOD1) in various subcellular locations. The molecular mechanism driving the formation of SOD1 aggregates is not fully understood but numerous studies suggest that aberrant aggregation escalates with folding instability of mutant apoSOD1. Recent advances on combining organelle-targeting therapies with the anti-aggregation capacity of chemical chaperones have successfully reduce the subcellular load of misfolded/aggregated SOD1 as well as their downstream anomalous cellular processes at low concentrations (micromolar range). Nevertheless, if such local aggregate reduction directly correlates with increased folding stability remains to be explored. To fill this gap, we synthesized and tested here the effect of 9 ER-, mitochondria- and lysosome-targeted chemical chaperones on the folding stability of truncated monomeric SOD1 (SOD1bar) mutants directed to those organelles. We found that compound ER-15 specifically increased the native state stability of ER-SOD1bar-A4V, while scaffold compound FDA-approved 4-phenylbutyric acid (PBA) decreased it. Furthermore, our results suggested that ER15 mechanism of action is distinct from that of PBA, opening new therapeutic perspectives of this novel chemical chaperone on ALS treatment.

Optimizing the Cell Painting assay for image-based profiling.

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years' experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105.

Superoxide Dismutase 1 Folding Stability as a Target for Molecular Tweezers in SOD1-Related Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation are hallmarks of many severe neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease. As a supramolecular ligand that binds to lysine and arginine residues, the molecular tweezer CLR01 was found to modify the aggregation pathway of disease-relevant proteins in vitro and in vivo with beneficial effects on toxicity. However, the molecular mechanisms of how tweezers exert these effects remain mainly unknown, hampering further drug development. Here, we investigate the modulation mechanism of unfolding and aggregation pathways of SOD1, which are involved in amyotrophic lateral sclerosis (ALS), by CLR01. Using a truncated version of the wildtype SOD1 protein, SOD1bar , we show that CLR01 acts on the first step of the aggregation pathway, the unfolding of the SOD1 monomer. CLR01 increases, by ∼10 °C, the melting temperatures of the A4V and G41D SOD1 mutants, which are commonly observed mutations in familial ALS. Molecular dynamics simulations and binding free energy calculations as well as native mass spectrometry and mutational studies allowed us to identify K61 and K92 as binding sites for the tweezers to mediate the stability increase. The data suggest that the modulation of SOD1 conformational stability is a promising target for future developments of supramolecular ligands against neurodegenerative diseases.

Cellular ATP Levels Determine the Stability of a Nucleotide Kinase.

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf 0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

The Unfolding Journey of Superoxide Dismutase 1 Barrels under Crowding: Atomistic Simulations Shed Light on Intermediate States and Their Interactions with Crowders.

The thermal stability of the superoxide dismutase 1 protein in a crowded solution is investigated by performing enhanced sampling molecular simulations. By complementing thermal unfolding experiments done close to physiological conditions (200 mg/mL), we provide evidence that the presence of the protein crowder bovine serum albumin in different packing states has only a minor, and essentially destabilizing, effect. The finding that quinary interactions counteract the pure stabilization contribution stemming from excluded volume is rationalized here by exploring the SOD1 unfolding mechanism in microscopic detail. In agreement with recent experiments, we unveil the importance of intermediate unfolded states as well as the correlation between protein conformations and local packing with the crowders. This link helps us to elucidate why certain SOD1 mutations involved in the ALS disease reverse the stability effect of the intracellular environment.

Protein Folding Modulation in Cells Subject to Differentiation and Stress.

Cytomimetic media are used to mimic the physicochemical properties of the cellular milieu in an in vitro experiment. The motivation is that compared to entire cells, they can be used efficiently in combination with a broad range of experimental techniques. However, the development and use of cytomimetic media is hampered by the lack of in-cell data that could be used as a hallmark to directly evaluate and improve the performance of cytomimetic media in different applications. Such data must include the study of specific biomolecular reactions in different cell types, different compartments of a single cells and different cellular conditions. In previous studies, model systems such as cancer cell lines, bacteria or oocytes were used. Here we studied how the environment of cells that undergo neuronal differentiation or proteostasis stress modulates the protein folding equilibrium. We found that NGF induced differentiation leads to a decrease of the melting temperature and a change of the folding mechanism. Proteomic changes that occur upon differentiation could explain this effect, however, we found that the crowding effect remained unchanged. Using MG132, a common proteasome inhibitor and inducer of the unfolded protein response, we show that changes to the quality control machinery modulate the folding equilibrium, leading to protein destabilization at prolonged stress exposure. Our study explores the range of protein folding modulation within cells subject to differentiation or stress that must be encountered in the development of cytomimetic media.

Effects of in vivo conditions on amyloid aggregation.

One of the grand challenges of biophysical chemistry is to understand the principles that govern protein misfolding and aggregation, which is a highly complex process that is sensitive to initial conditions, operates on a huge range of length- and timescales, and has products that range from protein dimers to macroscopic amyloid fibrils. Aberrant aggregation is associated with more than 25 diseases, which include Alzheimer's, Parkinson's, Huntington's, and type II diabetes. Amyloid aggregation has been extensively studied in the test tube, therefore under conditions that are far from physiological relevance. Hence, there is dire need to extend these investigations to in vivo conditions where amyloid formation is affected by a myriad of biochemical interactions. As a hallmark of neurodegenerative diseases, these interactions need to be understood in detail to develop novel therapeutic interventions, as millions of people globally suffer from neurodegenerative disorders and type II diabetes. The aim of this review is to document the progress in the research on amyloid formation from a physicochemical perspective with a special focus on the physiological factors influencing the aggregation of the amyloid-ß peptide, the islet amyloid polypeptide, α-synuclein, and the hungingtin protein.

Stability Effect of Quinary Interactions Reversed by Single Point Mutations.

In cells, proteins are embedded in a crowded environment that controls their properties via manifold avenues including weak protein-macromolecule interactions. A molecular level understanding of these quinary interactions and their contribution to protein stability, function, and localization in the cell is central to modern structural biology. Using a mutational analysis to quantify the energetic contributions of single amino acids to the stability of the ALS related protein superoxide dismutase I (SOD1) in mammalian cells, we show that quinary interactions destabilize SOD1 by a similar energetic offset for most of the mutants, but there are notable exceptions: Mutants that alter its surface properties can even lead to a stabilization of the protein in the cell as compared to the test tube. In conclusion, quinary interactions can amplify and even reverse the mutational response of proteins, being a key aspect in pathogenic protein misfolding and aggregation.

Imperfect crowding adaptation of mammalian cells towards osmotic stress and its modulation by osmolytes.

Changes of the extracellular milieu could affect cellular crowding. To prevent detrimental effects, cells use adaptation mechanisms to react to such conditions. Using fluorescent crowding sensors, we show that the initial response to osmotic stress is fast but imperfect, while the slow response renders cells more tolerant to stress, particularly in the presence of osmolytes.

Role of Electrostatics in Protein-RNA Binding: The Global vs the Local Energy Landscape.

U1A protein-stem loop 2 RNA association is a basic step in the assembly of the spliceosomal U1 small nuclear ribonucleoprotein. Long-range electrostatic interactions due to the positive charge of U1A are thought to provide high binding affinity for the negatively charged RNA. Short range interactions, such as hydrogen bonds and contacts between RNA bases and protein side chains, favor a specific binding site. Here, we propose that electrostatic interactions are as important as local contacts in biasing the protein-RNA energy landscape toward a specific binding site. We show by using molecular dynamics simulations that deletion of two long-range electrostatic interactions (K22Q and K50Q) leads to mutant-specific alternative RNA bound states. One of these states preserves short-range interactions with aromatic residues in the original binding site, while the other one does not. We test the computational prediction with experimental temperature-jump kinetics using a tryptophan probe in the U1A-RNA binding site. The two mutants show the distinct predicted kinetic behaviors. Thus, the stem loop 2 RNA has multiple binding sites on a rough RNA-protein binding landscape. We speculate that the rough protein-RNA binding landscape, when biased to different local minima by electrostatics, could be one way that protein-RNA interactions evolve toward new binding sites and novel function.

Inhibition of Huntingtin Exon-1 Aggregation by the Molecular Tweezer CLR01.

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.

Conformational dynamics and self-association of intrinsically disordered Huntingtin exon 1 in cells.

Huntington's disease is caused by a CAG trinucleotide expansion mutation in the Huntingtin gene that leads to an artificially long polyglutamine sequence in the Huntingtin protein. A key feature of the disease is the intracellular aggregation of the Huntingtin exon 1 protein (Httex1) into micrometer sized inclusion bodies. The aggregation process of Httex1 has been extensively studied in vitro, however, the crucial early events of nucleation and aggregation in the cell remain elusive. Here, we studied the conformational dynamics and self-association of Httex1 by in-cell experiments using laser-induced temperature jumps and analytical ultracentrifugation. Both short and long polyglutamine variants of Httex1 underwent an apparent temperature-induced conformational collapse. The temperature jumps generated a population of kinetically trapped species selectively for the longer polyglutamine variants of Httex1 proteins. Their occurrence correlated with the formation of inclusion bodies suggesting that such species trigger further self-association.

RNA Hairpin Folding in the Crowded Cell.

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin-structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high-molecular-weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.

The macromolecular crowding effect--from in vitro into the cell.

The influence of the cellular milieu, a complex and crowded solvent, is often neglected when biomolecular structure and function are studied in vitro. To mimic the cellular environment, crowding effects are commonly induced in vitro using artificial crowding agents like Ficoll or dextran. However, it is unclear if such effects are also observed in cellulo. Diverging results on protein stability in living cells point out the need for new quantitative methods to investigate the contributions of excluded volume and nonspecific interactions to the cellular crowding effect. We show how new crowding sensitive probes may be utilized to directly investigate crowding effects in living cells. Moreover, we discuss processes where crowding effects could play a crucial role in molecular cell biology.

Excluded-volume effects in living cells.

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions.