Biology subjects

Horowitz, S.

Publications and source records attributed to Horowitz, S..

12 recordsLinked to original sources

bioRxiv2026

Structural basis of chaperone mechanisms in cells and the evolutionary emergence of the protein world

How chaperones mediate protein folding in the crowded cell environment remains poorly understood. To gain insight, we developed CHAP-SEQ to examine how chaperones affect protein folding in cells at high throughput and amino acid resolution. Performing CHAP-SEQ using three chaperone proteins and one chaperone RNA reveals distinct modes of folding assistance. Chaperone proteins act preferentially on hydrophobic core residues, whereas chaperone RNA primarily targets structural or dynamic signatures. Furthermore, while the chaperone RNA has little preference for clients baseline foldability, the chaperone proteins favor clients with greater intrinsic foldability. These differences are consistent with an evolutionary hypothesis in which greater chaperone complexity played a role in the formation of stable hydrophobic cores, suggesting a potential link between chaperone function and the evolution of protein folding.

biochemistry↗

bioRxiv2025

G-quadruplex sequence dictates tau oligomerization and fibril morphology

Previous studies have identified that non-canonical nucleic acid structures known as G-quadruplexes (G4s) modulate protein aggregation and could play major roles in neurodegenerative diseases. Here we examine the presence and protein oligomerization activity of G4s that are enriched in human hippocampal aggregates. We found these G4s to be powerful, sequence-specific modulators of aggregation. Different G4s facilitated aggregate propagation in cells, caused tau to form distinct protein oligomer populations, and seeded tau fibrils with different fibril structure and length. This study highlights the importance of nucleic acid composition within aggregates, where small changes in nucleotide sequence and topology can vastly alter protein interactions, oligomerization, and fibrillization.

biochemistry↗

bioRxiv2025

Quantitative Thermodynamic Characterization of Self-Assembling RNA Nanostructures

Recent developments in RNA nanotechnology have led to the rise in designing specific higher order RNA structures with functional goals in mind, such as drug delivery and immunomodulation. As researchers create RNA nanostructures with the goal of becoming common in the molecular biologists toolkit, more investigation is required on the robustness of RNA designs. Primarily, what different molecular contexts are the designed and intended nanostructures stable in? In this work we show that by using second-order right-angle light scattering, that RNA nanostructure self-assembly is highly sensitive to environmental conditions. While a test RNA hexagonal grid nanostructure that forms correctly though 120{degrees} kissing loops under ideal conditions, small variations in salt conditions and annealing times cause the nanostructure to form less structured variants. Tertiary contacts for self-assembly require magnesium and break over a broad range of low temperatures, melting at 42 {degrees}C. In contrast this was found to be considerably lower than the secondary structure melting which occurred at 75 {degrees}C. This work underscores the importance of quantitative and thermodynamic characterization of self-assembling nanostructures as they begin to be deployed for engineering and therapeutic applications.x1

biochemistry↗

bioRxiv2024

G-quadruplexes catalyze protein folding by reshaping the energetic landscape

Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing G-quadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. As short nucleic acids can catalyze protein folding without the input of ATP, the ability of the cell to fold proteins is far higher than previously anticipated. Significance StatementHow folding of proteins occurs en masse in the cell is still a daunting unsolved problem. Many proteins have complicated and difficult folding trajectories, with in vitro folding times that are physiologically untenable. The acceleration of protein folding to physiologically relevant timescales is a biologically essential function thought to be accomplished by a small set of ATP-dependent chaperonins. In this work, we surprisingly show that small nucleic acid sequences containing G-quadruplexes can catalyze protein folding and reshape protein folding energy landscapes. As a result, the capacity for accelerating protein folding in the cells is far higher than previously suggested, potentially explaining the accommodation of large number of proteins with physiologically unreasonable folding times.

biophysics↗

bioRxiv2024

Reconstructing Biological Molecules with Help from Video Gamers

Foldit is a citizen science video game in which players tackle a variety of complex biochemistry puzzles. Here, we describe a new series of puzzles in which Foldit players improve the accuracy of the public repository of experimental protein structure models, the Protein Data Bank (PDB). Analyzing the results of these puzzles showed that the Foldit players were able to considerably improve the deposited structures and thus, in most cases, improved the output of the automated PDB-REDO refinement pipeline. These improved structures are now being hosted at PDB-REDO. These efforts highlight the continued need for the engagement of the lay population in science.

biophysics↗

bioRxiv2024

Manipulating TDP43 Aggregation via RNA G-quadruplexes

The events that lead to protein misfolding diseases are not fully understood. Many proteins implicated in neurodegenerative diseases (e.g., TDP43) interact with nucleic acids, including RNA G-quadruplexes. In this work, we investigate whether RNA G-quadruplexes play a role in TDP43 condensation in biophysical and cellular models. We find that G-quadruplexes modulate TDP43 aggregation in vitro and condensation in multiple cell types, including yeast, HEK293T, and motor-neuron-like NSC-34 cells. In yeast cells, treatment with G-quadruplexes causes increased TDP43 accumulation in cells before cellular death. In HEK293T cells expressing TDP43, incubation with G-quadruplex-binding small molecules causes an increase in G4 stability that also stabilizes TDP43 and reduces TDP43 condensation induced by proteasomal or oxidative stress. Finally, in NSC-34 cells overexpressing exogenous TDP43, we show that G-quadruplexes co-localize with TDP43 condensates under stress conditions and treatment with G-quadruplex-binding small molecules decreases TDP43-mediated toxicity. Together, these findings suggest that a novel future approach to explore for treating protein misfolding diseases may be to target specific RNA structures such as G-quadruplexes.

molecular biology↗

bioRxiv2023

A New Role for RNA G-quadruplexes in Aging and Alzheimer's Disease

INTRODUCTIONAs the world population ages, new molecular targets in aging and Alzheimers Disease (AD) are needed to combat the expected influx of new AD cases. Until now, the role of RNA structure in aging and neurodegeneration has largely remained unexplored. METHODSIn this study, we examined human hippocampal postmortem tissue for the formation of RNA G-quadruplexes (rG4s) in aging and AD. RESULTSWe found that rG4 immunostaining strongly increased in the hippocampus with both age and with AD severity. We further found that neurons with accumulation of phospho-tau immunostaining contained rG4s, that rG4 structure can drive tau aggregation, and that rG4 staining density depended on APOE genotype in the human tissue examined. DISCUSSIONCombined with previous studies showing the dependence of rG4 structure on stress and the extreme power of rG4s at oligomerizing proteins, we propose a model of neurodegeneration in which chronic rG4 formation is linked to proteostasis collapse. These morphological findings suggest that further investigation of RNA structure in neurodegeneration is a critical avenue for future treatments and diagnoses.

neuroscience↗

bioRxiv2022

G-quadruplexes rescuing protein folding

Maintaining the health of the proteome is a critical cellular task. Recently, we found G-quadruplex (G4) nucleic acids are especially potent at preventing protein aggregation in vitro and could at least indirectly improve the protein folding environment of E. coli. However, the roles of G4s in protein folding were not yet explored. Here, through in vitro protein folding experiments, we discover that G4s can accelerate protein folding by rescuing kinetically trapped intermediates to both native and near-native folded states. Time-course folding experiments in E. coli further demonstrate that these G4s primarily improve protein folding quality in E. coli as opposed to preventing protein aggregation. The ability for a short nucleic acid to rescue protein folding opens up the possibility of nucleic acids and ATP-independent chaperones to play considerable roles in dictating the ultimate folding fate of proteins.

biophysics↗

bioRxiv2022

Why are G-quadruplexes good at preventing protein aggregation?

Maintaining a healthy protein folding environment is essential for cellular function. Recently, we found that nucleic acids, and G-quadruplexes in particular, are potent chaperones for preventing protein aggregation. With the aid of structure-function and NMR analyses of two G-quadruplex forming sequences, PARP-I and LTR-III, we uncovered several contributing factors that affect G-quadruplexes in preventing protein aggregation. Notably, three factors emerged as vital in determining holdase activity of G-quadruplexes: their structural topology, structural dynamics, and oligomerization state. These factors together appear to largely dictate whether a G-quadruplex is able to prevent partially misfolded proteins from aggregating. Understanding the genesis of G-quadruplexes power as chaperones is an important facet to elucidating various protein aggregation diseases. Key Points- How nucleic acids act as protein chaperones is currently unknown. - G-quadruplexes are excellent at preventing protein aggregation, and here we describe basic tenets of this activity. - This activity could help design treatments for multiple neurodegenerative diseases.

biophysics↗

bioRxiv2021

Foldamers Reveal and Validate Novel Therapeutic Targets Associated with Toxic α-Synuclein Self-Assembly

Parkinsons disease (PD) is a progressive neurodegenerative disorder for which there is no successful prevention or intervention. The pathological hallmark for PD involves the self-assembly of functional Alpha-Synuclein (S) into non-functional amyloid structures. One of the potential therapeutic interventions against PD is the effective inhibition of S aggregation. However, the bottleneck towards achieving this goal is the identification of S domains/sequences that are essential for aggregation. Using a protein mimetic approach, we have identified S sequences-based novel targets that are essential for aggregation and will have significant therapeutic implications. An extensive array of in vitro, ex vivo, and in vivo assays was utilized to validate S sequences and their structural characteristics that are essential for aggregation and propagation of PD phenotypes. The study aids in developing significant mechanistic and therapeutic insights into various facets of S aggregation, which will pave the way for novel and effective treatments for PD.

biochemistry↗

bioRxiv2019

G-Quadruplexes Act as Sequence Dependent Chaperones via Protein Oligomerization

Maintaining proteome health is important for cell survival. Nucleic acids possess the ability to prevent aggregation up to 300-fold more efficiently than traditional chaperone proteins. In this study, we explore the sequence specificity of the chaperone activity of nucleic acids. Evaluating over 500 nucleic acid sequences effects on aggregation, we demonstrate that the holdase chaperone effect of nucleic acids is highly sequence dependent. Quadruplexes are found to have especially potent effects on aggregation with many different proteins via quadruplex:protein oligomerization. These observations contextualize recent reports of quadruplexes playing important roles in aggregation-related diseases, such as Fragile X and Amyotrophic lateral sclerosis (ALS).

biochemistry↗

bioRxiv2019

Identifying dynamic, partially occupied residues using anomalous scattering

X-ray crystallography is generally used to take single snapshots of a proteins conformation. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes Residual Anomalous and Electron Density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data collection and analysis strategy for partially occupied iodine anomalous signals. Using the long wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with as low as ~12% occupancy is demonstrated. The number and position of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly dynamic sections of crystal structures.\n\nSynopsisStructural studies on partially occupied, dynamic protein systems by crystallography are difficult. We present methods here for detecting these states in crystals.

biophysics↗