Biology subjects

Carrico, C.

Publications and source records attributed to Carrico, C..

6 recordsLinked to original sources

bioRxiv2026

A Multispecies, Modality-Agnostic Scalable In Vivo Mosaic Screening Platform for Therapeutic Target Discovery

Validating therapeutic targets for complex diseases requires investigating gene functions within native tissue architectures rather than reductionist in vitro models. Here we present a modality-agnostic AAV-based in vivo high-throughput screening platform capable of delivering knockouts, gain-of-function, and synthetic miRNA knockdowns directly to cells within the diseased environment. This system scales to hundreds of perturbations and is adaptable to diverse species and organ systems. To translate high-dimensional screen data into therapeutic assessment, we established a curated analysis framework that scores single-cell transcriptomes against human disease-specific molecular signatures. This method enables quantitative ranking of targets across distinct biological domains ranging from structural fibrosis to inflammatory signaling, to narrow in on the therapeutic potential of each intervention. We applied this strategy to screen loss- and gain-of-function libraries in a murine pulmonary fibrosis model and within the spontaneously osteoarthritic joints of aged horses, identifying metabolic, antifibrotic, and immunomodulatory targets. Importantly, our analysis framework successfully predicted functional outcomes in orthogonal human ex vivo tissue models, including soluble collagen reduction in lung slices and glycosaminoglycan restoration in cartilage, thus establishing a powerful paradigm for prioritizing therapeutic targets by uniting human disease signatures with highly multiplexed in vivo functional genomics.

systems biology↗

bioRxiv2025

Low Dose GLP-1 Therapy Attenuates Pathological Cardiac and Hepatic Remodelling in HFpEF Independent of Weight Loss

BACKGROUND AND AIMSHeart failure with preserved ejection fraction (HFpEF) remains a therapeutic challenge. GLP-1 receptor agonists (GLP-1RAs) show clinical promise, and the prevailing hypothesis is that their benefits are primarily driven by weight loss and the downstream benefits of improved functional status. We investigated the weight loss-independent effects of low-dose GLP-1RA therapy in a clinically relevant rodent model of severe cardiometabolic HFpEF. METHODSTen-week-old male ZSF1 obese rats with spontaneous HFpEF were treated with low-dose semaglutide (30 nmol/kg twice weekly, n=6) or vehicle for 16 weeks. Comprehensive assessments included body weight, 2-D echocardiography, invasive hemodynamics, exercise capacity as well as cardiac and hepatic fibrosis and lipid deposition. The study utilized advanced multi-omics approaches, including single-cell RNA sequencing of the heart and liver, as well as cardiac, hepatic and plasma proteomics, to explore underlying mechanisms. RESULTSIn ZSF1 obese rats, low-dose semaglutide in the absence of weight loss, significantly improved cardiac function, exercise tolerance, and attenuated fibrosis in the heart and liver. Interestingly, semaglutide therapy reduced cardiac and hepatic lipid content as well as lipid droplets in cardiac myocytes and hepatocytes. Mechanistically, multi-omics analyses of cardiac and hepatic tissues revealed that semaglutide exerted these benefits by improving cardiac metabolism, interfering with pro-fibrotic and pro-hypertrophic signals, and by reducing systemic inflammation. CONCLUSIONSLow-dose semaglutide provides significant cardioprotective, hepatoprotective, and metabolic benefits in HFpEF independent of weight loss. Our findings support the investigation of lower GLP-1RA dosing in HFpEF and other cardiovascular conditions, including in non-obese patients, to expand the clinical utility of these potent drugs. Translational PerspectiveWe demonstrate that low-dose semaglutide attenuates HFpEF-mediated pathological cardiac and hepatic remodelling in HFpEF independently of the weight loss effects of GLP-1 receptor activation. Primary mechanisms are attenuated cardiac and hepatic fibrosis and reverse lipid transport. These findings provide a mechanistic basis for the direct cardiovascular actions of GLP-1RAs, revealing their potential to modulate key disease drivers like fibrosis and lipotoxicity. These data support the use of lower, better-tolerated doses of GLP-1RAs to treat HFpEF, potentially benefiting a wider range of patients, including those who are not obese or who suffer from side effects with current GLP-1 regimens.

pharmacology and toxicology↗

bioRxiv2022

In-depth Analysis of the Sirtuin 5-regulated Mouse Brain Acylome using Library-free Data-Independent Acquisitions

Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry-based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data-independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach that combines antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock-out mice, and discovered and quantified 466 malonylated and 2,211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 171 malonylated and 640 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome. Statement of significance of the studyPost-translational modifications (PTMs) are key regulators of protein structure, functions, and interactions. A great variety of PTMs have been discovered, including lysine acylation, such as acetylation, malonylation, and succinylation. Lysine acylation is understudied, particularly in the brain, and analysis by mass spectrometry-based proteomics faces significant challenges. In this study, we present a robust and efficient workflow to investigate proteome-wide PTM remodeling combining affinity PTM enrichment and a novel spectral library-free data-independent acquisition (DIA) approach. The strength of label-free DIA becomes evident with the collection of comprehensive information by tandem mass spectrometry for all detectable precursor ions of all biological samples, and the highly accurate quantitative information that can subsequently be retrieved with time-efficient and straightforward library-free strategies. More importantly, this enables confident identification of PTM sites and differentiation of PTM isomers. We applied this workflow to decipher the malonylome and succinylome remodeling and cross-talk in brains from wild-type and Sirt5(-/-) mice, taking advantage of the demalonylase and desuccinylase activities of SIRT5, a nicotinamide adenine dinucleotide (NAD+)-dependent sirtuin. Interestingly, 10 malonylated proteins and 33 succinylated proteins targeted by SIRT5 are involved in the Parkinsons disease pathway, including subunit beta of the calcium/calmodulin-dependent protein kinase type II (Camk2b) and protein DJ-1 (Park7).

cell biology↗

bioRxiv2022

Regulation of urea cycle by reversible high stoichiometry lysine succinylation

The post-translational modification, lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. In the present study, using stable isotope labeling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in urea cycle, are regulated by SIRT5. Mutation of the high stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance and reduces locomotor and exploratory activity in male mice upon high-ammonium diet feeding. Therefore, lysine succinylation is functionally important in ammonia metabolism.

molecular biology↗

bioRxiv2022

Histone malonylation is regulated by SIRT5 and KAT2A

The posttranslational modification lysine malonylation is found in many proteins, including histones. However, it remains unclear whether histone malonylation is regulated or functionally relevant. Here, we report that availability of malonyl-co-enzyme A (malonyl-CoA), an endogenous provider of malonyl groups, affects lysine malonylation, and that the deacylase SIRT5 selectively reduces malonylation of histones. To determine if histone malonylation is enzymatically catalyzed, we knocked down each of the 22 lysine acetyltransferases (KATs) to test their malonyltransferase potential. KAT2A knockdown in particular reduced histone malonylation levels. By mass spectrometry, H2B_K5 was highly malonylated and significantly regulated by SIRT5 in mouse brain and liver. Acetyl-CoA carboxylase (ACC), the malonyl-CoA producing enzyme, was partly localized in the nucleolus, and histone malonylation increased nucleolar area and ribosomal RNA expression. Levels of global lysine malonylation and ACC expression were higher in older mouse brains than younger mice. These experiments highlight the role of histone malonylation in ribosomal gene expression.

molecular biology↗

bioRxiv2022

Coenzyme A binding sites induce proximal acylation across protein families

Lysine N{varepsilon}-acylations, such as acetylation or succinylation, are post-translational modifications that regulate protein function. In mitochondria, lysine acylation is predominantly non-enzymatic, and only a specific subset of the proteome is acylated. Coenzyme A (CoA) can act as an acyl group carrier via a thioester bond, but what controls the acylation of mitochondrial lysines remains poorly understood. Using published datasets, here we found that proteins with a CoA-binding site are more likely to be acetylated, succinylated, and glutarylated. Using computational modeling, we show that lysine residues near the CoA-binding pocket are highly acylated compared to those farther away. We hypothesized that acyl-CoA binding enhances acylation of nearby lysine residues. To test this hypothesis, we co-incubated enoyl-CoA hydratase short chain 1 (ECHS1), a CoA-binding mitochondrial protein, with succinyl-CoA and CoA. Using mass spectrometry, we found that succinyl-CoA induced widespread lysine succinylation and that CoA competitively inhibited ECHS1 succinylation. CoA-induced inhibition at a particular lysine site correlated inversely with the distance between that lysine and the CoA-binding pocket. Our study indicated that CoA acts as a competitive inhibitor of ECHS1 succinylation by binding to the CoA-binding pocket. Together, this suggests that proximal acylation at CoA-binding sites is a primary mechanism for lysine acylation in the mitochondria.

biochemistry↗