Biology subjects

Stein, A.

Publications and source records attributed to Stein, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Glutamatergic and Redox Abnormalities in Major Depressive Disorder: Links to Childhood Maltreatment and Perceived Stress

Background: Childhood sexual abuse (CSA) increases the risk for major depressive disorder (MDD), yet the underlying neurobiological mechanisms remain poorly understood. We examined (1) associations of medial prefrontal cortex (mPFC) glutamatergic metabolites and redox balance with childhood maltreatment and MDD; and (2) whether these neurochemical measures predicted future perceived stress. Methods: Using proton and phosphorus 4T magnetic resonance spectroscopy, we quantified glutamate, glutamine, their sum (Glx), and the NAD/NADH ratio within the same mPFC voxel in 71 women across four groups: healthy controls (HC; n=27), current MDD without CSA (n=14), current MDD with CSA (n=14), and remitted MDD with CSA (n=16). Results: Glutamate and Glx levels were lower in both CSA-exposed groups (n=30) compared with HC (n=27), whereas women with MDD without CSA did not differ significantly from HC. Among CSA-exposed women, greater maltreatment severity was associated with lower glutamine, and greater maltreatment multiplicity with lower glutamate and Glx, specifically in women with current MDD. The association between depressive symptom severity and glutamine differed by CSA history. A significant interaction between baseline NAD/NADH ratio and clinical group predicted perceived stress at 6-month follow-up, adjusting for baseline perceived stress. Specifically, lower baseline NAD/NADH ratio predicted greater subsequent perceived stress in MDD without CSA, but not in the other clinical groups. Conclusions: Our findings suggest that mPFC glutamatergic metabolites and redox balance show distinct patterns of association with CSA history, depression status, and subsequent perceived stress, highlighting neurochemical heterogeneity in MDD and molecular markers of risk.

neuroscience↗

bioRxiv2026

Chromosomal instability shapes spatial and temporal phenotypic diversity in a malignant peripheral nerve sheath tumour

Understanding the spatial and temporal dynamics of tumour evolution is crucial for determining the drivers of cancer progression. Linking genotype to phenotype across a tumour remains challenging, however. Here, we use single-cell and spatial multi-omics to comprehensively profile a primary malignant peripheral nerve sheath tumour (MPNST) and its multifocal recurrence. Combining the native barcoding system from extensive heterogeneity in copy number alterations with mutation data, we resolve the evolutionary tree of this tumour, revealing a branching structure suggestive of ongoing chromosomal instability. We show that gene dosage effects contribute to phenotypic diversity and scale predominantly linearly with copy number. Using spatial genomics assisted by laser capture microdissection and spatial transcriptomics, we performed in situ lineage tracing in this human tumour, elucidating the relationship between local expansions and interactions between tumour cells and the microenvironment. These findings demonstrate the potential of combined bulk, single-cell and spatial techniques to dissect cancer evolution in time and space and link genotype to phenotype in detail.

genomics↗

bioRxiv2026

An Atlas of Short Linear Motif-Mediated Human Protein-Protein Interactions

Short linear motifs (SLiMs) within intrinsically disordered protein regions mediate transient interactions crucial for cell physiology1. However, the global interaction landscape of human SLiMs remains largely uncharted. Here we present the Atlas of SLiM-mediated Human protein-protein Interactions (ASHI), which maps more than 20,000 interactions by screening over 800 human protein domains against a library of one million peptides tiling the human disordered proteome. ASHI expands the SLiM interactome, uncovers novel binding modes for known peptide-binding domains, and reveals unexpected peptide-binding activities in enzymes, chaperones, RNA-binding proteins, and modification-reader domains. Furthermore, intrinsically disordered regions emerge as densely encoded interaction platforms where interaction specificity is governed by diverse mechanisms, including key motif determinants, flanking residues, competition, and multivalency. These data provide an unprecedented foundation for modeling dynamic interaction networks, interpreting disease-associated variants, and decoding the dark proteome.

biochemistry↗

bioRxiv2025

Blocking protein quality control degradation leads to structural stabilization of DHFR indel variants

Gene variants leading to insertions or deletions of amino acid residues (indels) often have detrimental consequences for the folding of the encoded protein. Yet at some positions indels are tolerated or result merely in partial unfolding and hypomorphic phenotypes. Typically unfolded proteins are targeted for protein quality control (PQC) degradation via the ubiquitin-proteasome system, which in yeast is mediated by specific E3 ubiquitin-protein ligases, including Ubr1 and San1. Here we systematically probed the folding of a library of indel variants in the DHFR protein using a sensitive yeast-based protein folding reporter. We show that deletion of Ubr1 or San1 leads to a greater fraction of folded DHFR indel variants, primarily positioned towards the N- and C-terminal regions in DHFR. Intriguingly, most of the DHFR indels that are structurally stabilized in the E3 knockout strains, are also stabilized at lowered temperatures and upon binding the DHFR inhibitor methotrexate. This suggests that blocking PQC degradation can restore function to partially unfolded hypomorph variants, thus providing a potential therapeutic avenue for protein misfolding diseases.

biochemistry↗

bioRxiv2025

Muscle Fiber- and Cell Type-Specificity of Training Adaptation in Male Mice

Skeletal muscle possesses extraordinary plasticity of structure, metabolism, and function in response to repeated contractile activity. As a syncytium embedded within a complex microenvironment, muscle relies on the coordination of distinct myonuclear populations and diverse mononucleated cell types. Here, we present a high-resolution single-nucleus RNA-sequencing atlas of 550000 skeletal muscle nuclei, capturing the longitudinal transcriptional responses of 17 distinct myonuclear and 21 mononuclear cell populations at multiple time points after one bout of exhaustive exercise in trained and sedentary mice. The transcriptional programs of these populations are further shaped by training status into divergent adaptive trajectories. A subset of oxidative myonuclei enters a delayed regenerative state post-exercise, reflecting a bifurcated response to a disproportionate metabolic load on fibers during endurance exercise. Prior training accelerates homeostatic recovery and shields oxidative nuclei from exacerbated damage signatures. In parallel, mononucleated cells emerge as the primary mediators of intercellular communication during recovery. Together, this dataset establishes that training adaptation emerges through a coordinated interplay of intrinsic adaptive programs of multicellular remodeling, and provides a foundational resource for mechanistic insights into muscle plasticity.

physiology↗

bioRxiv2025

Framework for combined functional and computational assessment of variant pathogenicity in the sodium leak channel NALCN

The sodium leak channel nonselective (NALCN) is the pore-forming channel component of the NALCN channelosome. This protein complex generates a depolarizing sodium leak in various tissues and contributes to respiratory and circadian rhythms, locomotion, and sleep. De novo and inherited variants of NALCN can lead to severe developmental syndromes called contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD) and infantile hypotonia with psychomotor retardation and characteristic facies 1 (IHPRF1), respectively. Although variants of uncertain significance (VUS) or presumed pathogenic variants have been studied in heterologous expression systems before, there is no generally accepted framework on how to assess or predict variant pathogenicity. We set out use the functional and computational characterization of 19 VUS detected in CLIFAHDD and IHPRF1 patients to establish a robust analysis to classify suspected disease-causing variants. Specifically, we employ a combination of multiple parameters derived from two-electrode voltage-clamp electrophysiology recordings and predicted protein stability and conservation scores. We show that this approach is capable of distinguishing benign common variants from both gain- and loss-of-function (GoF/LoF) variants. Additionally, our work provides mechanistic insight into the molecular mechanism underlying specific variants and provides insight into the unusual propensity of NALCN missense variants to result in GoF phenotypes. We anticipate that this experimental and computational framework will aid assessment of variant pathogenicity of NALCN and other components of the channelosome in the future.

genetics↗

bioRxiv2025

UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation

Protein-lipid crosstalk is fundamental to homeostasis in the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway, a branch of the ubiquitin-proteasome system, maintains ER membrane properties by degrading lipid metabolic enzymes. However, the ERAD components that sense membrane properties and their mechanisms remain poorly defined. Using reconstituted systems with purified ERAD factors, we show that membrane composition modulates the ubiquitination cascade at multiple levels. The membrane-anchored E2 UBE2J2 acts as a sensor for lipid packing: in loosely packed membranes, UBE2J2 becomes inactive due to membrane association that impedes ubiquitin loading, while tighter packing promotes its active conformation and interaction with E1. UBE2J2 activity directs ubiquitin transfer by the E3 ligases RNF145, MARCHF6, and RNF139, targeting both themselves and the substrate squalene monooxygenase. Additionally, RNF145 senses cholesterol, altering its oligomerization and activity. These findings reveal that ERAD integrates multiple lipid signals, with UBE2J2 relaying and extending the effect of lipid signals through its cooperation with multiple E3 ligases.

biochemistry↗

bioRxiv2025

Targeted Irradiation and STAT3 Inhibition Reprogram the AML Microenvironment and Extend Survival: Toward Translational Immunoradiotherapy

PurposeDespite advances in acute myeloid leukemia (AML) therapy, relapses remain challenging. While AML is radiation-sensitive, total body irradiation (TBI) causes organ toxicities and activates tolerogenic/proangiogenic STAT3 signaling. CSI-2, a myeloid cell-targeted STAT3 inhibitor, promotes anti-leukemic immune responses but has limited efficacy against high disease burden. We investigated whether image-guided targeted marrow irradiation (TMI), which focuses radiation on leukemia sites while sparing critical organs, could synergize with CSI-2 to improve leukemia clearance and establish durable immunity. MethodsMice were intravenously engrafted with CMM-AML cells reaching 20-30% in bone marrow (BM) infiltration (moderate-to-high disease burden) before receiving IV-injections of CSI-2 (5mg/kg) with or without TMI. Fluorescently labeled CSI-2 biodistribution was assessed using flow cytometry and quantitative multiphoton microscopy. Survival was monitored for 3-4 months before evaluating BM composition using flow cytometry and immunohistochemistry. ResultsTMI significantly improved vascular permeability and scavenger receptor/TLR9-dependent uptake of CSI-2 by AML cells and leukemia-associated myeloid cells. Combined TMI/CSI-2 treatment more effectively reduced high leukemia burden than CSI-2 alone, achieving >80% survival at 120 days with increased CD8+ cytotoxic and CD4+ helper T cell infiltration. TMI/CSI-2-treated mice were protected from AML rechallenge suggesting that they developed protective immune memory. In an aggressive MLL-AF9 AML model, TMI/CSI-2 combination significantly extended survival compared to either monotherapy. ConclusionTMI/CSI-2 strategy represents a novel organ-sparing immunoradiotherapy that synergistically enhances leukemia clearance while promoting long-term protective immunity. These findings warrant further investigation of this strategy for high-burden or relapsed AML and provide the foundation for clinical translation.

immunology↗

bioRxiv2025

A complete map of human cytosolic degrons and their relevance for disease

Degrons are short protein segments that target proteins for degradation via the ubiquitin-proteasome system and thus ensure timely removal of signaling proteins and clearance of misfolded proteins from the intracellular space. Here, we describe a systematic screen for degrons in the human cytosol. We determine degron potency of >200,000 different 30-residue tiles from more than 5,000 cytosolic human proteins with 99.7% coverage. In total, 19.1% of the tiles function as strong degrons, 30.4% as intermediate degrons, while 50.5% did not display degron properties. The vast majority of the degrons are dependent on the E1 ubiquitin-activating enzyme and the proteasome but independent of autophagy. The results reveal both known and novel degron motifs, both internal as well as at the C-terminus. Mapping the degrons onto protein structures, predicted by AlphaFold2, revealed that most of the degrons are located in buried regions, indicating that they only become active upon unfolding or misfolding. Training of a machine learning model allowed us to probe the degron properties further and predict the cellular abundance of missense variants that operate by forming degrons in exposed and disordered protein regions, thus providing a mechanism of pathogenicity for germline coding variants at such positions.

biochemistry↗

bioRxiv2025

Brain-derived synaptic vesicles have an intrinsic ability to sequester tubulin

The presence and function of microtubules within the synaptic bouton has long been under investigation. In recent years evidence has accumulated that connects the synaptic vesicle cluster to the local dynamics of microtubule ends. Nonetheless, one question remains open, namely whether the vesicles influence the availability of tubulin within the synaptic compartment. An analysis of previously published live imaging experiments indicates that tubulin is strongly enriched in the synaptic vesicle cluster. To analyze the vesicle-tubulin interaction directly, we isolated vesicles from the mouse brain and imaged them together with fluorescent tubulin in vitro. We found that soluble tubulin is collected by synaptic vesicles in physiological buffers, resulting in the formation of tubulin-rich regions (TRRs) on the respective vesicle clusters. We conclude that the synaptic vesicle cluster is indeed able to recruit soluble tubulin.

neuroscience↗

bioRxiv2025

Genomic in vitro transcription and Nanopore direct RNA sequencing of a human B-Lymphocyte cell line

Genomic DNA used as a template for in vitro transcription of RNA can serve as a true negative control for benchmarking RNA modification detection by Nanopore direct RNA sequencing (DRS) models. We generated DRS data for in vitro transcribed (IVT) RNA composed of canonical nucleotides using genomic DNA from a human cell line. We applied Dorado modification calling models to these data, and calculated 9-mer specific false-positive rates for eight RNA modifications as a comparison point for future development of RNA modification models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/650674v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@962feorg.highwire.dtl.DTLVardef@4237aborg.highwire.dtl.DTLVardef@154d346org.highwire.dtl.DTLVardef@1fad8f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

On the patterns of genetic intra-tumour heterogeneity before and after treatment

Genetic intra-tumour heterogeneity (gITH) is a universal property of all cancers. It emerges from the interplay of cell division, mutation accumulation and selection with important implications for the evolution of treatment resistance. Theoretical and data-driven approaches extensively studied gITH in ageing somatic tissues or cancers at detection. Yet, the expected patterns of gITH during and after treatment are less well understood. Here, we use stochastic birth-death processes to investigate the expected patterns of gITH across different treatment scenarios. We consider homogeneous treatment response with shrinking, growing and stable disease, and follow up investigating heterogeneous treatment response with sensitive and resistant cell types. We derive analytic expressions for the site frequency spectrum, the total mutational burden and the single-cell mutational burden distribution that we validate with computer simulations. We find that the SFS after homogeneous treatment response retains its characteristic power-law tail, while emergent resistant clones cause peaks corresponding to their sizes. The frequency of the largest resistant clone is subdominant and independent of the population size at detection, whereas the relative total number of resistant cells increases with detection size. Furthermore, the growth dynamics under treatment determine whether the total mutational burden is dominated by preexisting or newly acquired mutations, suggesting different possible treatment strategies.

biophysics↗

bioRxiv2024

A high-speed OLED monitor for precise stimulation in vision, eye-tracking, and EEG research

The recent introduction of organic light-emitting diode (OLED) monitors with refresh rates of 240 Hz or more opens new possibilities for their use as precise stimulation devices in vision research, experimental psychology, and electrophysiology. These affordable high-speed monitors, targeted at video gamers, promise several advantages over the cathode ray tube (CRT) and liquid crystal display (LCD) monitors commonly used in these fields. Unlike LCDs, OLED displays have self-emitting pixels that can show true black, resulting in superior contrast ratios, a broad color gamut, and good viewing angles. More importantly, the latest gaming OLEDs promise excellent timing properties with minimal input lags and rapid transition times. However, OLED technology also has potential drawbacks, notably Auto-Brightness Limiting (ABL) behavior, where the local luminance of a stimulus can change with the number of currently illuminated pixels. This study characterized a 240 Hz OLED monitor, the ASUS PG27AQDM, in terms of its timing properties, spatial uniformity, viewing angles, warm-up times, and ABL behavior. We also compared its responses to those of CRTs and LCDs. Results confirm the monitors excellent temporal properties with CRT-like transition times (around 0.3 ms), wide viewing angles, and decent spatial uniformity. Additionally, we found that ABL could be prevented with appropriate settings. We illustrate the monitors benefits in two time-critical paradigms: Rapid "invisible" flicker stimulation and the gaze-contingent presentation of stimuli during eye movements. Ourfindings suggest that the newest gaming OLEDs are precise and cost-effective stimulation devices for visual experiments that have several key advantages over CRTs and LCDs.

neuroscience↗

bioRxiv2024

Disentangling the mutational effects on protein stability and interaction of human MLH1

Missense mutations can have diverse effects on proteins, depending on their location within the protein and the specific amino acid substitution. Mutations in the DNA mismatch repair gene MLH1 are associated with Lynch syndrome, yet the underlying mechanism of most disease-causing mutations remains elusive. To address this gap, we aim to disentangle the mutational effects on two essential properties for MLH1 function: protein stability and protein-protein interaction. We systematically examine the cellular abundance and interaction with PMS2 of 4839 (94%) MLH1 variants in the C-terminal domain. Our combined data shows that most MLH1 variants lose interaction with PMS2 due to reduced cellular abundance. However, substitutions to charged residues in the canonical interface lead to reduced interaction with PMS2. Unexpectedly, we also identify a distal region in the C-terminal domain of MLH1 where substitutions cause both decreased and increased binding with PMS2, and propose a region in PMS2 as the binding site. Our data successfully distinguish benign from pathogenic MLH1 variants and correlate with thermodynamic stability predictions and evolutionary conservation. This work provides mechanistic insights into variant consequences and may help interpret MLH1 variants. Author SummaryMutations in proteins, including single amino acid substitutions, can disrupt normal cellular function and lead to diseases. Hence, there is great interest in understanding their consequences from both basic science and clinical genetics perspectives. Loss-of-function variants in the human mismatch repair protein MLH1 are associated with Lynch syndrome, a hereditary condition that significantly increases the risk of certain cancers. Detailed insights into the molecular consequences of MLH1 variants are essential for understanding the mechanisms underlying Lynch syndrome. In this study, we use high-throughput experimental approaches to test how missense mutations affect the cellular abundance of MLH1 and the interaction with its partner, PMS2. We find that many variants disrupting the interaction with PMS2 also lead to reduced cellular abundance levels, suggesting a strong relationship between protein stability and interaction in MLH1. Additionally, we identify a potential novel interaction site between MLH1 and PMS2, where mutations in MLH1 either increase or decrease interaction without affecting abundance. Our datasets are in line with the classification of clinically annotated MLH1 variants and may aid in interpreting variants of uncertain significance, while also providing new insights into the molecular details of the interaction with PMS2.

molecular biology↗

bioRxiv2024

Systematic characterization of indel variants using a yeast-based protein folding sensor

Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet compared to missense variants the effects of indels are poorly understood and predicted. To approach this issue, we developed a sensitive protein folding sensor based on complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor accurately reports on the folding of disease-linked missense variants and de novo designed proteins. Applying the folding sensor to a saturated library of single amino acid indel variants in human DHFR revealed that most regions which tolerate indels are confined to internal loops and the N- and C-termini. Surprisingly, indels are also allowed at a central -helix. Several indels are temperature-sensitive and the folding of most of these indels is rescued upon binding to the competitive DHFR inhibitor methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels operate by destabilizing the native fold and that these computational tools may be useful for classification of indels observed in population sequencing.

biochemistry↗

bioRxiv2023

A joint embedding of protein sequence and structure enables robust variant effect predictions

The ability to predict how amino acid changes may affect protein function has a wide range of applications including in disease variant classification and protein engineering. Many existing methods focus on learning from patterns found in either protein sequences or protein structures. Here, we present a method for integrating information from protein sequences and structures in a single model that we term SSEmb (Sequence Structure Embedding). SSEmb combines a graph representation for the protein structure with a transformer model for processing multiple sequence alignments, and we show that by integrating both types of information we obtain a variant effect prediction model that is more robust to cases where sequence information is scarce. Furthermore, we find that SSEmb learns embeddings of the sequence and structural properties that are useful for other downstream tasks. We exemplify this by training a downstream model to predict protein-protein binding sites at high accuracy using only the SSEmb embeddings as input. We envisage that SSEmb may be useful both for zero-shot predictions of variant effects and as a representation for predicting protein properties that depend on protein sequence and structure.

bioinformatics↗

bioRxiv2023

Selective sweep probabilities in spatially expanding populations

Evolution during range expansions shapes biological systems from microbial communities and tumours up to invasive species. A fundamental question is whether, when a beneficial mutation arises during a range expansion, it will evade clonal interference and sweep through the population to fixation. However, most theoretical investigations of range expansions have been confined to regimes in which selective sweeps are effectively impossible, while studies of selective sweeps have either assumed constant population size or have ignored spatial structure. Here we use mathematical modelling and analysis to investigate selective sweep probabilities in the alternative yet biologically relevant scenario in which mutants can outcompete and displace a slowly spreading wildtype. Assuming constant radial expansion speed, we derive probability distributions for the arrival time and location of the first surviving mutant and hence find surprisingly simple approximate and exact expressions for selective sweep probabilities in one, two and three dimensions, which are independent of mutation rate. Namely, the selective sweep probability is approximately (1 - cwt/cm)d, where cwt and cm are the wildtype and mutant radial expansion speeds, and d the spatial dimension. Using agent-based simulations, we show that our analytical results accurately predict selective sweep frequencies in the two-dimensional spatial Moran process. We further compare our results with those obtained for alternative growth laws. Parameterizing our model for human tumours, we find that selective sweeps are predicted to be rare except during very early solid tumour growth, thus providing a general, pan-cancer explanation for findings from recent sequencing studies.

evolutionary biology↗

bioRxiv2023

Deep mutational scanning reveals a tight correlation between protein degradation and toxicity of thousands of non-native aspartoacylase protein variants

When the structural stability of a protein is compromised, the protein may form non-native interactions with other cell proteins and thus becomes a hazard to the cell. To mitigate this danger, destabilized proteins are targeted by the cellular protein quality control (PQC) network, which either corrects the folding defect or targets the protein for degradation. However, the details of how the protein folding and degradation systems collaborate to combat potentially toxic non-native proteins are unknown. To address this issue, we performed systematic studies on destabilized variants of the cytosolic aspartoacylase, ASPA, where loss-of-function variants are linked to Canavans disease, an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. Using Variant Abundance by Massively Parallel sequencing (VAMP-seq), we determined the abundance of 6152 out of the 6260 ([~]98%) possible single-site missense and nonsense ASPA variants in cultured human cells. The majority of the low abundance ASPA variants are degraded through the ubiquitin-proteasome system (UPS) and become toxic upon prolonged expression. Variant cellular abundance data correlates with predicted thermodynamic stability, evolutionary conservation, and separates most known disease-linked variants from benign variants. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in ASPA are located in buried regions, and reveals that the wild-type ASPA C-terminal region functions as a degron. Collectively, our data can be used to interpret Canavans disease variants and also offer mechanistic insight into how ASPA missense variants are targeted by the PQC system. These are essential steps towards future implementation of precision medicine for Canavans disease.

molecular biology↗