Biology subjects

Ramos, D.

Publications and source records attributed to Ramos, D..

11 recordsLinked to original sources

bioRxiv2026

The structural basis for LRRK2's activation and autoinhibition

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the second most common cause of autosomal-dominant Parkinsons disease (PD), and increased LRRK2 kinase activity is also observed in idiopathic PD, making LRRK2 a major actionable therapeutic target. LRRK2 is a 286-kDa multidomain enzyme containing a Ras-like GTPase (ROC) and a kinase domain. Using cryo-EM, biochemical reconstitution, and cell-based assays, we show that the ROC GTPase governs switching between autoinhibited and active states: GTP binding promotes activation, whereas GDP binding enforces autoinhibition. Two common PD-linked mutations, G2019S and R1441C/G/H, activate LRRK2 through distinct structural mechanisms, revealing genotype-specific routes to dysregulation. These findings provide a unified framework for understanding LRRK2 regulation with broad therapeutic implications. Stabilizing the GDP-bound state may inhibit LRRK2 by maintaining autoinhibition, whereas promoting the GTP-bound state could be advantageous in specific cellular contexts, such as the lung, where increased LRRK2 kinase activity may play protective or regulatory roles.

neuroscience↗

bioRxiv2025

Mechanism of Lutein to meso-Zeaxanthin Isomerization by RPE65 Catalysis

The macular pigments lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) protect the human retina from light and oxidative stress. While L and Z are abundant in the human diet, MZ is nearly absent. We previously demonstrated MZ is derived from L in chicken embryos precisely timed with expression of RPE65. Herein, we show that RPE65 from mouse, an animal that does not concentrate MZ in the eye, catalyzes L to MZ isomerization similarly as found for RPE65 from chicken and human, when expressed in cultured cells. Co-expression with xanthophyll-binding proteins had no impact on MZ yield. L and MZ both fit deep into the tunnel accessing the non-heme iron center, with strain evident for the 3R,6R {varepsilon} ring of L. Interestingly, a negatively charged Glu148, found along substrate tunnel, highly conserved among carotenoid cleavage dioxygenases, and which is critical for eye health, could be replaced by a neutral, isosteric residue (Gln) without impacting MZ yield. We propose that L to MZ isomerization proceeds by a neutral, radical transition state that differs from the carbocation encountered during retinoid isomerization. These findings extend our mechanistic understanding for macular carotenoid metabolism and should be considered when developing therapeutic interventions that act via RPE65.

biochemistry↗

bioRxiv2025

A Cryptic Binding Pocket Regulates the Metal-Dependent Activity of Cas9

Cas9 is a metal-dependent nuclease that has revolutionized gene editing across diverse cells and organisms exhibiting varying ion uptake, metabolism, and concentrations. However, how divalent metals impact its catalytic function, and consequently its editing efficiency in different cells, remains unclear. Here, extensive molecular simulations, Markov State Models, biochemical and NMR experiments, demonstrate that divalent metals - Mg2+, Ca2+, and Co2+ - promote activation of the catalytic HNH domain by binding within a dynamically forming divalent metal binding pocket (DBP) at the HNH-RuvC interface. Mutations in DBP residues disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying this cryptic DBP as a key regulator of Cas9s metal-dependent activity. The ionic strength thereby promotes Cas9s conformational activation, while its catalytic activity is metal-specific. These findings are critical to improving the metal-dependent function of Cas9 and its use for genome editing in different cells and organisms.

biophysics↗

bioRxiv2025

Prevention of Transgene Silencing During Human PluripotentStem Cell Differentiation

While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occurs with both randomly integrated transgenes, introduced via transposase or lentiviral methods, and with transgenes targeted to specific genomic sites, including at commonly used safe harbor loci. The challenge to robustly express experimental transgenes in differentiated pluripotent stem cells is a major bottleneck in the field for applications such as CRISPR screening. Here, we conducted a comparative analysis to systematically evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of promoters and transcriptional stability elements are able to prevent transgene silencing during differentiation, whereas chromatin insulators had less impact on silencing and three novel safe harbor integration sites performed similarly to the CLYBL locus. Guided by these insights we developed the PiggyBac vector TK4, which showed complete resistance to transgene silencing across various neuronal and microglial differentiation protocols from six different pluripotent stem cell lines, as independently confirmed by seven different laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation, and holds the potential for broad applications in various research fields.

neuroscience↗

bioRxiv2025

A preclinical pig model of Angelman syndrome mirrors the early developmental trajectory of the human condition

Angelman syndrome is a neurodevelopmental disorder characterized by severe motor and cognitive deficits. It is caused by the loss of the maternally inherited allele of the imprinted ubiquitin-protein ligase E3A (UBE3A) gene. Rodent models of Angelman syndrome do not fully recapitulate all the symptoms associated with the condition and are limited as a preclinical model for therapeutic development. Here, we show that pigs (Sus scrofa) with a maternally inherited deletion of UBE3A (UBE3A-/+) have altered postnatal behaviors, impaired vocalizations, reduced brain growth, motor incoordination, and ataxia. Neonatal UBE3A-/+ pigs exhibited several symptoms observed in infants with Angelman syndrome, including hypotonia, suckling deficits, and failure to thrive. Collectively, these findings are consistent with the pathophysiology and developmental trajectory observed in individuals with Angelman syndrome. We anticipate that this pig model will advance our understanding of the pathophysiology of Angelman syndrome and be used as a preclinical large animal model for therapeutic development.

genetics↗

bioRxiv2023

Rapid species discovery and identification with real-time barcoding facilitated by ONTbarcoder 2.0 and Oxford Nanopore R10.4

Most arthropod species are undescribed and hidden in biodiversity samples that are difficult to sort to species using morphological characters. Sorting specimens to putative species with DNA barcodes is an attractive alternative, but needs cost-effective techniques that are suitable for use in many laboratories around the world. Barcoding with Oxford Nanopores portable and inexpensive MinION sequencers could be a good presorting approach because it reduces the space and capital cost of a fully functional barcoding laboratory. Similarly important would be user-friendly and reliable software for analysis of the ONT data. It is here provided in the form of an updated ONTbarcoder that is available for all commonly used operating systems and includes a GUI. The new version of ONTbarcoder has three key improvements related to the higher read quality obtained with ONTs latest basecallers and chemistry (R10.4 flow cells, Q20+ kits). Firstly, ONTbarcoder can now deliver real-time barcoding to complement ONTs real-time sequencing. This means that the first barcodes are obtained within minutes of starting a sequencing run and a significant proportion of barcodes are available before sequencing completes. This real-time feature of ONTbarcoder can be configured to handle both Mk1B and Mk1C sequencers. The input is a demultiplexing sheet and sequencing data (raw or basecalled). Secondly, the improved read quality of ONTs latest flow cells (R10.4) allows for the use of primers with shorter indices than those previously needed (9 bp vs. 12-13 bp). This decreases primer cost and improves PCR success rates. Thirdly, we demonstrate that the availability of R10.4 chemistry in the low-cost Flongle flow cell is an attractive option for users who require only 200-250 barcodes at a time.

ecology↗

bioRxiv2023

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development. One Sentence SummaryLoss of TDP-43 function results in the expression of de novo proteins from mis-spliced mRNA transcripts.

neuroscience↗

bioRxiv2022

Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade

Cascades are RNA-guided multi-subunit CRISPR-Cas surveillances complexes that target foreign nucleic acids for destruction. Here, we present a 2.9-[A] resolution cryo-electron (cryo-EM) structure of the D. vulgaris type I-C Cascade bound to a double-stranded (ds)DNA target. Our data shows how the 5-TTC-3 protospacer adjacent motif (PAM) sequence is recognized, and provides a unique mechanism through which the displaced, single-stranded non-target strand (NTS) is stabilized via stacking interactions with protein subunits in order to favor R-loop formation and prevent dsDNA re-annealing. Additionally, we provide structural insights into how diverse anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking initial DNA binding. These observations provide a structural basis for directional R-loop formation and reveal how divergent Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.

biochemistry↗

bioRxiv2021

A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

neuroscience↗

bioRxiv2021

Inhibition of CDK4/6 Overcomes Primary Resistance to PD-1 Blockade in Malignant Mesothelioma

BackgroundDespite the profound number of malignant pleural mesothelioma (MPM) patients now treated with PD-1 blockade, insight into the underpinnings of rational therapeutic strategies to treat resistance to checkpoint immunotherapy remain unrealized. Our objective was to develop a novel therapeutic approach to overcome primary resistance to PD-1 blockade in MPM. MethodsWe generated a transcriptome signature of resistance to PD-1 blockade in MPM patients treated with nivolumab (4 responders and 4 non-responders). We used the TCGA MPM cohort (N=73) to determine what genomic alterations were associated with the resistance signature. We tested whether regulation of identified molecules could overcome resistance to PD-1 blockade in an immunocompetent mouse malignant mesothelioma model. ResultsImmunogenomic analysis by applying our anti-PD-1 resistance signature to the TCGA cohort revealed that deletion of CDKN2A was highly associated with primary resistance to PD-1 blockade. Under the hypothesis that resistance to PD-1 blockade can be overcome by CDK4/6 inhibition, we tested whether CDK4/6 inhibitors could overcome resistance to PD-1 blockade in subcutaneous tumors derived from Cdkn2a(-/-) AB1 malignant mesothelioma cells, which were resistant to PD-1 blockade. The combination of daily oral administration of CDK4/6 inhibitors (abemaciclib or palbociclib) and intraperitoneal anti-PD-1 treatment markedly suppressed tumor growth, compared with anti-PD-1 or CDK4/6 inhibitor alone. ConclusionsWe identified a novel therapeutic target, CDK4/6, to overcome primary resistance to PD-1 blockade through comprehensive immunogenomic approaches. These data provide a rationale for undertaking clinical trials of CDK4/6 inhibitors in the more than 40% of patients with MPM who demonstrate loss of CDKN2A.

immunology↗

bioRxiv2019

Oxytocin Neurons Enable Social Transmission of Maternal Behavior

Maternal care is profoundly important for mammalian survival, and non-biological parents can express it after experience with infants. One critical molecular signal for maternal behavior is oxytocin, a hormone centrally released by hypothalamic paraventricular nucleus (PVN). Oxytocin enables plasticity within the auditory cortex, a necessary step for responding to infant vocalizations. To determine how this change occurs during natural experience, we continuously monitored homecage behavior of female virgin mice co-housed for days with an experienced mother and litter, synchronized with recordings from virgin PVN cells, including from oxytocin neurons. Mothers engaged virgins in maternal care by ensuring their nest presence, and demonstrated maternal behavior in self-generated pup retrieval episodes. These social interactions activated virgin PVN and gated behaviorally-relevant cortical plasticity for pup vocalizations. Thus rodents can acquire maternal behavior by social transmission, and our results describe a mechanism for adapting brains of adult caregivers to infant needs via endogenous oxytocin. One Sentence SummaryMother mice help co-housed virgins become maternal by enacting specific behaviors that activate virgin oxytocin neurons.

neuroscience↗