Biology subjects

Meller, A.

Publications and source records attributed to Meller, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Chaperone isoform and interactome mapping reveals functional diversification of DNAJA2-DNAJA4 complexes via stress-regulated isoforms

The human HSP70 chaperone network maintains cellular proteostasis through a diverse repertoire of HSP70s and co-chaperones. Here we examine alternative isoforms and co-chaperone hetero-complexes as additional sources of network complexity. To that end, we systematically mapped the isoform, tissue-expression, and interaction landscapes of the human HSP70 network, revealing a modular interactome containing known and novel DNAJ-DNAJ interactions. We found both tissue-expression isoform divergence as well as widespread alternative-canonical isoform co-expression, suggesting additional modes of functional diversification. Focusing on the uncharacterized DNAJA2-DNAJA4 hetero-complex, we identified the stress-inducible isoform DNAJA4-CTD-II. DNAJA4-CTD-II formed hetero-complexes with DNAJA2 and DNAJA4, with both interactions enhanced following sodium arsenite stress. Functionally, DNAJA4-CTD-II co-localized with TDP-43 aggregates and significantly suppressed their accumulation in a DNAJA2-dependent manner. Together, our data reveal extensive, uncharted isoform and interaction complexity within the HSP70 network, and uncover isoform-dependent hetero-complex remodeling as a new layer of chaperone network regulation.

systems biology↗

bioRxiv2024

Targeting FUS-ALS aggregation with Proteasome Inhibitors

ALS, Amyotrophic lateral sclerosis, a devastating neurodegenerative disease (ND) with no cure, is often caused by abnormal cytosolic aggregation of RNA-binding proteins, the most well-known of which are TDP-43 and FUS. The proteasome is considered one of the major systems that degrades misfolded, including ND-associated, proteins, thereby acting to reduce aggregation, while inhibition of the proteasome increases aggregation. Unexpectedly, we found that proteasome inhibitor treatment significantly reduced ALS-associated mutant FUS aggregation in cells and in primary neurons. This is in sharp contrast to most other ND-associated aggregating proteins, including Huntingtin and TDP-43, for which proteasome inhibitors enhanced aggregation. We further found that this inhibitory effect is dependent on the transcription factor HSF1, suggesting that the underlying mechanism of this effect is transcriptionally-mediated. Since heat shock treatment did not show any effect on FUS aggregation, we hypothesized that proteasome inhibitors elicit a transcriptional program distinct of that of heat shock, which is protective of FUS aggregation. We identified BAG3, a co-chaperone that cooperates with HSP70 in reducing FUS aggregation, as a significant mediator of this effect. We therefore propose BBB-permeable proteasome inhibitors as a potential therapy specific to ALS-FUS.

molecular biology↗

bioRxiv2024

The Pseudogene RPS27AP5 Reveals Novel Ubiquitin and Ribosomal Protein Variants Involved in Specialised Ribosomal Functions.

Pseudogenes, traditionally considered non-functional gene copies resulting from evolutionary mutations, have garnered attention due to recent transcriptomics and proteomics revealing their unexpected expressions and consequential cellular functions. Ubiquitin, transcribed from UBA52 and RPS27A genes, fused to ribosomal proteins eL40 and eS31, and polyubiquitin precursors encoded by UBB and UBC genes, has additional pseudogenes labeled as non-functional. However, recent evidence challenges this notion, demonstrating that these pseudogenes produce ubiquitin variants with minimal differences from the canonical sequence, suggesting a new regulatory dimension in ubiquitin-mediated cellular processes. To systematically catalogue possible Ubiquitin (Ub) and Ubiquitin-like (Ubl) variants from pseudogenes, expression data was compiled, identifying potential functional variants. Among these pseudogenes, RPS27AP5 expresses both Ubiquitin variant (UbP5) and ribosomal protein variant (S27aP5), with precursor proteins maturing through cleavage and exhibiting behavior similar to their counterparts post-translation. Notably, S27aP5 integrates into translating ribosomes, increasing the 80S monosomal ribosomal fraction and indirectly influencing p16INK4A transcriptional activation. The discovery of a functional S27a pseudogene supports the concept that a subset of ribosomes may incorporate diverse subunits for specific translational functions.

cell biology↗

bioRxiv2023

Single protein molecules separation, tracking and counting in ultra-thin silicon channels

Emerging single-molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra-fast full-size protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms and low throughputs. Here, we introduce a single-molecule method for parallel protein separation and tracking, yielding multi-dimensional molecular properties used for their identification. Proteins are tagged by dual amino-acid specific labels and are electrophoretically separated by their mass/charge in custom-designed silicon nano-channel. This approach allows us to analyze thousands of individual proteins within a few minutes by tracking their motion during the migration. We demonstrate the power of the method by quantifying a cytokine panel for host-response discrimination between viral and bacterial infections. Moreover, we show that two clinically-relevant splice isoforms of VEGF can be accurately quantified from human serum samples. Being non-destructive and compatible with full-length intact proteins, this method opens up new ways for antibody-free single protein molecule quantification.

biophysics↗

bioRxiv2023

Divergent Molecular Phenotypes in Point Mutations at the Same Residue in Beta-Myosin Heavy Chain Lead to Distinct Cardiomyopathies

In genetic cardiomyopathies, a frequently described phenomenon is how similar mutations in one protein can lead to discrete clinical phenotypes. One example is illustrated by two mutations in beta myosin heavy chain ({beta}-MHC) that are linked to hypertrophic cardiomyopathy (HCM) (Ile467Val, I467V) and left ventricular non-compaction (LVNC) (Ile467Thr, I467T). To investigate how these missense mutations lead to independent diseases, we studied the molecular effects of each mutation using recombinant human {beta}-MHC Subfragment 1 (S1) in vitro assays. Both HCM-I467V and LVNC-I467T S1 mutations exhibited similar mechanochemical functions, including unchanged ATPase and enhanced actin velocity but had distinct effects on the basal activity of myosin. HCM-I467V S1 showed no change in basal ATPase activity of myosin while LVNC-I467T reduced the basal ATPase activity by 50%. Molecular dynamics simulations reveal that I467T allosterically disrupts nucleotide binding of myosin, which may contribute to the uncoupled reduced basal activity and enhanced actin velocity observed in this mutation. These contrasting molecular effects may lead to contractile dysregulation that initiates LVNC-associated signaling pathways that progress the phenotype. Together, analysis of these mutations provides evidence that phenotypic complexity originates at the molecular level and is critical to understanding disease progression and developing therapies.

biophysics↗

bioRxiv2023

Homologous mutations in β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in {beta}-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus- pseudocamptodactyly syndrome. It is not known if their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human {beta}, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins, with the most dramatic in perinatal, but minimal effects in {beta} myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing ATPase cycle rate. In contrast, the only measured effect of R671C in {beta} myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not {beta}, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents the first direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are yet another testament to myosins highly allosteric nature.

biophysics↗

bioRxiv2023

Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors

Virtual screening is a widely used tool for drug discovery, but its predictive power can vary dramatically depending on how much structural data is available. In the best case, crystal structures of a ligand-bound protein can help find more potent ligands. However, virtual screens tend to be less predictive when only ligand-free crystal structures are available, and even less predictive if a homology model or other predicted structure must be used. Here, we explore the possibility that this situation can be improved by better accounting for protein dynamics, as simulations started from a single structure have a reasonable chance of sampling nearby structures that are more compatible with ligand binding. As a specific example, we consider the cancer drug target PPM1D/Wip1 phosphatase, a protein that lacks crystal structures. High-throughput screens have led to the discovery of several allosteric inhibitors of PPM1D, but their binding mode remains unknown. To enable further drug discovery efforts, we assessed the predictive power of an AlphaFold-predicted structure of PPM1D and a Markov state model (MSM) built from molecular dynamics simulations initiated from that structure. Our simulations reveal a cryptic pocket at the interface between two important structural elements, the flap and hinge regions. Using deep learning to predict the pose quality of each docked compound for the active site and cryptic pocket suggests that the inhibitors strongly prefer binding to the cryptic pocket, consistent with their allosteric effect. The predicted affinities for the dynamically uncovered cryptic pocket also recapitulate the relative potencies of the compounds ({tau}b=0.70) better than the predicted affinities for the static AlphaFold-predicted structure ({tau}b=0.42). Taken together, these results suggest that targeting the cryptic pocket is a good strategy for drugging PPM1D and, more generally, that conformations selected from simulation can improve virtual screening when limited structural data is available.

biophysics↗

bioRxiv2023

CRISPR/Cas9-mediated knockout of the ubiquitin variant UbKEKS reveals a role in regulating nucleolar structures and composition.

Ubiquitination is a post-translational modification responsible for one of the most complex multi-layered communication and regulation system in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Among them, the recently discovered ubiquitin variant UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. The absence of UbKEKS induces accumulation of lamin A in the nucleoli, highlighting the need for deeper investigations about protein composition and functions regulation of this highly dynamic and membrane-less compartment. By using data independent acquisition mass spectrometry and microscopy, we show here that despite not impacting protein stability, UbKEKS is required to maintain normal nucleolar organization. The absence of UbKEKS increases nucleolis size and accentuate their circularity while disrupting dense fibrillar component and fibrillar center structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Notably, lack of UbKEKS favors nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis in UbKEKS knockout cells observed by flow cytometry and real-time cellular growth monitoring. Overall, the results presented here identifies the first cellular functions of the UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the already complex ubiquitination system.

cell biology↗

bioRxiv2022

Novel subthreshold retinal laser treatment with ERG-based thermal dosimetry activates hormetic heat response in pig RPE in vivo

Boosting natural mechanisms to retain cellular homeostasis and combat oxidative stress by inducing a hormetic heat shock to retinal pigment epithelium with non-damaging transpupillary laser heating, i.e., with subthreshold laser treatment (SLT), has been suggested as a promising therapeutic target for many retinal diseases, including age-related macular degeneration. However, the therapeutic temperature window for the treatments is narrow and thermal dosimetry is unavailable. Here we introduce an SLT modality where the retinal temperature is monitored with electroretinography (ERG)-based thermal dosimetry and demonstrate its feasibility with anesthetized pigs. In 60-second treatments with 810 nm laser, the ED50 peak temperature for visible lesion generation was 48{degrees}C and the relative temperature determination error was below 10% from the temperature increase. Heat shock protein expression increased, and autophagy was activated at 44.2 {degrees}C and no signs of oxidative stress or apoptosis emerged at 44.2 {degrees}C or 46.5 {degrees}C. The demonstrated method permits a controlled activation of intracellular chaperones and waste clearance in RPE cell with a clear temperature margin for adverse events. In the clinical setting, ERG-based dosimetry would allow safe and personalized SLTs for retinal diseases currently lacking effective treatments.

bioengineering↗

bioRxiv2022

Accelerating cryptic pocket discovery using AlphaFold

Cryptic pockets, or pockets absent in ligand-free, experimentally determined structures, hold great potential as drug targets. However, cryptic pocket opening is often beyond the reach of conventional biomolecular simulations because certain cryptic pocket openings involve slow motions. Here, we investigate whether AlphaFold can be used to accelerate cryptic pocket discovery either by generating structures with open pockets directly or generating structures with partially open pockets that can be used as starting points for simulations. We use AlphaFold to generate ensembles for 10 known cryptic pocket examples, including 5 that were deposited after AlphaFolds training data was extracted from the PDB. We find that in 6 out of 10 cases AlphaFold samples the open state. For plasmepsin II, an aspartic protease from the causative agent of malaria, AlphaFold only captures partial pocket opening. As a result, we ran simulations from an ensemble of AlphaFold-generated structures and show that this strategy samples cryptic pocket opening, even though an equivalent amount of simulations launched from a ligand-free experimental structure fails to do so. Markov state models (MSMs) constructed from the AlphaFold-seeded simulations quickly yield a free energy landscape of cryptic pocket opening that is in good agreement with the same landscape generated with well-tempered metadynamics. Taken together, our results demonstrate that AlphaFold has a useful role to play in cryptic pocket discovery but that many cryptic pockets may remain difficult to sample using AlphaFold alone.

biochemistry↗

bioRxiv2022

Decoupling of mRNA and protein expression in aging brains reveals the age-dependent adaptation of specific gene subsets

During aging, changes in gene expression are associated with decline in physical and cognitive abilities. Here, we investigated the connection between changes of mRNA and protein expression in the brain by comparing the transcriptome and proteome of the mouse cortex during aging. Our transcriptomic analysis revealed that aging mainly triggers gene activation in the cortex. We showed that increase of mRNA expression correlates with protein expression, specifically in the anterior cingulate cortex where we also observed an increase of cortical thickness during aging. Genes exhibiting an aging-dependent increase of mRNA and protein levels are involved in sensory perception and immune functions. Our proteomic analysis also identified changes in protein abundance in the aging cortex and highlighted a subset of proteins that were differentially enriched but exhibited stable mRNA levels during aging, implying the contribution of aging-related post transcriptional and post-translational mechanisms. These specific genes were associated with general biological processes such as translation, ribosome assembly and protein degradation, but also important brain functions related to neuroplasticity. By decoupling mRNA and protein expression, we have thus characterized distinct subsets of genes that differentially adjust to cellular aging in the cerebral cortex.

biochemistry↗

bioRxiv2022

Drug specificity and affinity are encoded in the probability of cryptic pocket opening in myosin motor domains

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least 6 of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 milliseconds of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatins binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). To test our ability to make blind predictions, we predicted blebbistatins binding affinity for an isoform (Myh7b) whose blebbistatin sensitivity was unknown. Encouragingly, we find good agreement between the predicted and measured IC50 (0.67 {micro}M vs. 0.36 {micro}M). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets. SignificanceDrug development requires the discovery of compounds which specifically target one member of a protein family without triggering side effects that arise from interactions with other related proteins. Myosins are a family of motor proteins that are drug targets for heart diseases, cancer, and parasitic infections. Here, we investigate why the compound blebbistatin specifically inhibits some myosins more potently than others, even though its binding site is closed in all known experimental structures. We find that the blebbistatin binding pocket opens in molecular dynamics simulations of certain myosin motors, and that the probability of opening predicts how potently blebbistatin inhibits a particular motor. Our work suggests that differences in cryptic pocket formation can be exploited to develop specific therapeutics.

biophysics↗

bioRxiv2022

Predicting the locations of cryptic pockets from single protein structures using the PocketMiner graph neural network

Cryptic pockets expand the scope of drug discovery by enabling targeting of proteins currently considered undruggable because they lack pockets in their ground state structures. However, identifying cryptic pockets is labor-intensive and slow. The ability to accurately and rapidly predict if and where cryptic pockets are likely to form from a protein structure would greatly accelerate the search for druggable pockets. Here, we present PocketMiner, a graph neural network trained to predict where pockets are likely to open in molecular dynamics simulations. Applying PocketMiner to single structures from a newly-curated dataset of 39 experimentally-confirmed cryptic pockets demonstrates that it accurately identifies cryptic pockets (ROC-AUC: 0.87) >1,000-fold faster than existing methods. We apply PocketMiner across the human proteome and show that predicted pockets open in simulations, suggesting that over half of proteins thought to lack pockets based on available structures are likely to contain cryptic pockets, vastly expanding the druggable proteome.

biophysics↗

bioRxiv2022

Stress-induced transcriptional readthrough into neighboring genes is linked to intron retention

Exposure to certain stresses leads to readthrough transcription. Here we found that readthrough transcription often proceeds into the proximal gene downstream, in a phenomenon termed "read-in". Using polyA-selected RNA-seq data from mouse fibroblasts, we identified widespread read-in in heat shock, oxidative and osmotic stress conditions. We found that read-in genes share distinctive genomic characteristics; they are extremely short, and highly GC rich. Furthermore, using ribosome footprint profiling we found that translation of read-in genes is significantly reduced. Strikingly, read-in genes show extremely high levels of intron retention during stress, mostly in their first intron, which is not explained by features usually associated with intron retention, such as short introns and high GC content. Finally, we found that first introns in read-in genes have weaker splice sites. Our data portray a relationship between read-in and intron retention, suggesting it may have co-evolved to facilitate reduced translation of read-in genes during stress.

genomics↗

bioRxiv2020

Citizen Scientists Create an Exascale Computer to Combat COVID-19

SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression, and replication, which depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate an unprecedented 0.1 seconds of the viral proteome. Our simulations capture dramatic opening of the apo Spike complex, far beyond that seen experimentally, which explains and successfully predicts the existence of cryptic epitopes. Different Spike homologues modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also observe dramatic conformational changes across the proteome, which reveal over 50 cryptic pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.

biophysics↗

bioRxiv2019

Structural dynamics of isolated myosin motor domains encode differences in their mechanochemical cycles

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for twelve myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally-measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships. Subject AreasBiochemistry and Chemical Biology, Structural Biology and Molecular Physics

biophysics↗

bioRxiv2019

Fluorescent polysome profiling reveals stress-mediated regulation of HSPA14-ribosome interactions

Ribosome-associated factors play important roles in regulation of translation in response to various physiological and environmental signals. Here we present fluorescent polysome profiling, a new method that provides simultaneous detection of UV and fluorescence directly from polysome gradients. We demonstrate the capabilities of the method by following the polysome incorporation of different fluorescently tagged ribosomal proteins in human cells. Furthermore, we used fluorescent polysome profiling to examine chaperone-ribosome interactions, and characterized their changes in response to proteotoxic stress conditions. We revealed dynamic regulation of HSPA14-polysome association in response to heat shock, with a marked heat shock-mediated increase in the polysome-association of HSPA14. Our data suggest a model whereby HSPA14 dimerization is increased upon heat shock. We therefore established fluorescent polysome profiling as a streamlined method, that can enhance and facilitate the broad exploration of ribosome-associated factors and their regulation.

molecular biology↗

bioRxiv2019

Cellular proteostasis decline in human senescence

Proteostasis collapse, the diminished ability to maintain protein homeostasis, has been established as a hallmark of nematode aging. However, whether proteostasis collapse occurs in humans has remained unclear. Here we demonstrate that proteostasis decline is intrinsic to human senescence. Using transcriptome-wide characterization of gene expression, splicing and translation, we found a significant deterioration in the transcriptional activation of the heat shock response in stressed senescent cells. Furthermore, phosphorylated HSF1 nuclear localization and distribution were impaired in senescence. Interestingly, alternative splicing regulation was also dampened. Surprisingly, we found a decoupling between different Unfolded Protein Response (UPR) branches in stressed senescent cells. While young cells initiated UPR-related translational and transcriptional regulatory responses, senescent cells showed enhanced translational regulation and ER stress sensing, however they were unable to trigger UPR-related transcriptional responses. This was accompanied by diminished ATF6 nuclear localization in stressed senescent cells. Finally, we revealed a deterioration of proteasome function in senescence following heat stress, which did not recover upon return to normal temperature. Together, our data unraveled a deterioration in the ability to mount dynamic stress transcriptional programs upon human senescence with broad implications on proteostasis control, and connected proteostasis decline to human aging. SignificanceProtein homeostasis (proteostasis), the balance between protein synthesis, folding, and degradation, is thought to deteriorate with age, and the prevalence of protein misfolding diseases, e.g. Alzheimers, Parkinsons etc., with human aging is increased. However, while in worms this phenomenon has been well established, in humans it remained unclear. Here we show that proteostasis is declined in human senescence, i.e. cellular aging. We found that while stress sensing is enhanced in senescent cells, and their response at the level of protein synthesis is intact, they fail to properly activate multiple programs required for stress adaptation at the level of gene transcription. Our findings support the notion that proteostasis decline may have major implications on human aging.

molecular biology↗