Biology subjects

Estrada, M.

Publications and source records attributed to Estrada, M..

6 recordsLinked to original sources

bioRxiv2026

Synthetic expansion of non-coding DNA reveals physiological constraints on genome size

Genome size varies widely across eukaryotes, largely because of differences in non-coding DNA, but the physiological consequences of this variation remain unclear. To directly test how non-coding DNA abundance influences cellular physiology, we engineered a scalable genome-expansion system in the budding yeast S. cerevisiae that increases genome size while leaving the endogenous genome unchanged. By sequentially fusing yeast artificial chromosomes (YACs) carrying predominantly non-coding human DNA, we generated strains with up to 12.8 Mb of additional DNA, approximately doubling the native genome. Genome expansion reduced growth rate and increased cell size in proportion to the amount of non-coding DNA. Spike-in-normalized RNA-seq and ChIP-seq revealed that the non-coding DNA is pervasively transcribed, with a proportional amount of RNA polymerase II being redistributed from the endogenous genome to the added non-coding sequences. This resulted in a global decrease in the endogenous mRNA concentration. However, ribosome profiling and proteomics experiments revealed that there is little translation of YAC-associated transcripts. Our mathematical model shows that cellular growth rate decreases because non-coding DNA acts as a sink for transcriptional resources to lower the concentration of endogenous mRNA. Thus, our work links genome expansion to proliferative capacity and offers a mechanistic explanation for why the fastest-growing cells, such as yeast and bacteria, carry so little non-coding DNA.

Synthetic Biology↗

bioRxiv2025

Proteome-Wide Discovery of Degradable Proteins Using Bifunctional Molecules

Targeted protein degradation (TPD) is an emergent therapeutic strategy with the potential to circumvent challenges associated with targets unamenable to conventional pharmacological inhibition. Among TPD approaches, Proteolysis Targeting Chimeras (PROTACs) have shown marked advancement with numerous candidates in clinical development. Despite their potential, most PROTACs utilize advanced small molecule inhibitors, inherently limiting the scope of this approach. More generally, the fraction of the proteome tractable to PROTAC-type strategies is unknown. Here, we describe a chemical proteomic strategy for the agnostic discovery of degradable human proteins in cells using a new class of bifunctional degrader molecules called "AgnoTACs". Proteome-wide screening of 72 AgnoTACs in human cells uncovered downregulation events spanning >50 functionally and structurally diverse proteins, most of which lack chemical probes. Our findings highlight the potential of function-biased chemical libraries coupled with proteomic profiling to discover degrader starting points as well as furnish a blueprint for expanding our understanding of the chemically degradable proteome.

biochemistry↗

bioRxiv2025

Distinct cis-acting elements mediate co-localization of mRNAs encoding for co-translational interactors in cytoplasmic clusters

Many newly synthesized proteins assemble co-translationally, providing a vital mechanism to prevent subunit misfolding in the crowded cytoplasm. Initial evidence indicates that the spatial organization of mRNAs aids this assembly, but it is unclear how these mRNAs are organized and how common this mechanism is. We used single-molecule Fluorescence in situ Hybridization in Saccharomyces cerevisiae to examine the spatial organization of mRNAs encoding subunits of various cytosolic complexes involved in critical cellular functions, such as fatty acid synthesis, glycolysis, translation and various amino acid biosynthesis. We found that mRNAs of the same protein complex often co-localize in specific cytoplasmic clusters. Additionally, we observed that the mRNAs encoding enzymes of biosynthetic pathways are organized in cytosolic clusters. Focusing on mRNAs encoding fatty acid synthase complex subunits, we discovered that non-coding cis elements significantly influence mRNA localization in an additive manner. Specifically, 5 and 3 UTRs, together with further upstream and downstream regions, facilitate co-localization. Inhibiting mRNA co-localization impaired growth when complex activity was essential, highlighting the importance of mRNA spatial organization for cellular survival. Transiently disrupting mRNA translation also affected clustering, indicating that both the nascent chains and mRNA sequence targeting cues are combinatorically contributing to spatial organization. Proteomics analysis demonstrates the impact of cis-elements on the abundance of the encoded subunits, as well as the entire pathway. In summary, we provide evidence that mRNA co-localization in cytoplasmic foci is coordinated by complementary mechanisms crucial for co-translational assembly, allowing efficient regulation of protein complex formation and entire pathways.

cell biology↗

bioRxiv2023

Tiny Earth CURE improves student persistence in science

Course-based undergraduate research experiences (CUREs) enhance student retention in STEM, particularly among students who belong to historically excluded communities. Yet the mechanisms by which CUREs contribute to student integration and persistence are poorly understood. Utilizing the Tripartite Integration Model of Social Influence (TIMSI), this longitudinal study examines how Tiny Earth, an antibiotic-discovery CURE, impacts students scientific self-efficacy, scientific identity, endorsement of scientific community values, and intentions to persist in science. The study also explores how gains in TIMSI factors vary as a function of student demographics and course characteristics. Results of pre-and post-course measurements show that scientific self-efficacy and identity increased among students in Tiny Earth, and some student demographics and course characteristics moderated these gains. Gains in scientific self-efficacy, identity, and values correlated with gains in persistence intentions, whereas student demographics and course characteristics did not. Results of this study show that the Tiny Earth CURE enhanced students integration into the scientific community, which was linked to intentions of students of both historically underrepresented and majority groups to persist in STEM. We discuss how courses that provide opportunity to learn science skills in the context of a CURE can contribute toward enlarging and diversifying the STEM workforce.

scientific communication and education↗

bioRxiv2020

Multiple known and a novel parvovirus associated with an outbreak of feline diarrhea and vomiting

An unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and PCR. We characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by 8/17 affected cats and different feline bocaviruses shed by 9/17 cats. Also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. Epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease.

microbiology↗

bioRxiv2019

Protein Quality Control is a Risk Factor and Therapeutic Target in Toxin-Induced Biliary Atresia

BACKGROUND and AIMS Extra-hepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA.METHODS We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screen of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model with subsequent validation.RESULTS Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors (PDE5i) and other cGMP signaling activators worked synergistically with the glutathione precursor N- acetylcysteine (NAC) in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. PDE5i enhanced proteasomal degradation and required intact HSP90 chaperone.CONCLUSION Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone, and mirrors recently reported BA risk stratification linked to glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with NAC and cGMP signaling enhancers warrants further investigation as therapy for BA.Background and Context Biliary atresia (BA) is an obstructive fibrosing cholangiopathy that is the leading indication for liver transplantation in the pediatric population. There are no known treatments to prevent progressive liver injury after surgical restoration of bile flow.New Findings The authors identify factors that affect susceptibility of cholangiocytes to oxidative injury using a toxin-induced BA model. This information is used to validate genetic risk factors for human BA and identified PDE5i as a potential treatment for biliary atresia, either on its own or in combination with the anti-oxidant N-acetyl-cysteine.Limitations The work done in animal and cell culture models needs further study in human tissue-derived models and a larger cohort of BA patients.Impact The findings from this study provide a rationale for identifying new genetic risk factors that predispose to BA and for an interventional study to prevent progressive liver injury in this enigmatic disease.Short Summary This study uses zebrafish and human cell culture models to identify novel injury mechanisms, genetic risk factors and new therapies for the pediatric liver disease biliary atresia.Competing Interest StatementThe authors have declared no competing interest.Abbreviations17-AAG7-N-allylamino-17-demethoxygeldanamycin;BAbiliary atresia;bilbiliatresone;8-Br-cGMP8-Bromoguanosine 3’,5’-cyclic monophosphate;EHCextrahepatic cholangiocytes;Gclglutamate cysteine ligase;GGAgeranylgeranyl acetate;gsrglutathione reductase;GSHreduced glutathione;GSSGoxidized glutathione;IHCintrahepatic cholangiocytes;NACN-acetylcysteine;PDE5iPhosphodiesterase-5 inhibitors;PQCprotein quality control;WTwild-typeView Full Text

pathology↗