Biology subjects

Greco, T. M.

Publications and source records attributed to Greco, T. M..

10 recordsLinked to original sources

bioRxiv2026

Targeted quantification assays for DNA repair and handling proteins and interactions in Huntington's disease models

Huntingtons disease (HD) is a life-altering genetic neurodegenerative disorder, with cognitive, motor, and psycho-social effects that have consequential impacts on the individuals and their families. While current treatments improve disease symptoms, there are no FDA-approved therapies that prevent disease progression. Converging lines of evidence from human GWAS and mouse models point to DNA repair and handling (R/H) proteins as promising therapeutic targets due to their ability to modulate somatic expansion of the CAG repeat of HTT. The roles of DNA R/H HD modulator proteins are incompletely understood, in part, due to their relatively low cellular abundance and technical challenges in quantification. Here, we developed and validated targeted mass spectrometry assays quantifying DNA R/H proteins, spanning functions in mismatch repair, Fanconi anemia, and transcriptional regulation, using complementary workflows for timsTOF and Orbitrap platforms. We built species-specific experiment spectral libraries that outperformed in silico libraries for target detection. Applying this pipeline to an HTT-Q140 knock-in mouse HD model, we observed that DNA R/H protein abundances were largely unchanged in HD mice, while HTT and HAP40 showed increased nuclear association with disease progression. To facilitate translational research applications, we further developed a stable isotope dilution assay for absolute quantification of 11 human mismatch repair-associated proteins and generated an HTT knock-out human neuroblastoma cell line. Additionally, we used thermal proximity coaggregation profiling to characterize the endogenous interactomes of MMR proteins. We observed that HTT KO caused proteome down-regulation in selected DNA R/H proteins and reshaped the MMR protein interactome, with the most pronounced changes observed for MLH1 and PMS1 interactions. Overall, we established a validated, transferable assay for quantifying DNA R/H proteins in perturbation studies using human and mouse HD model systems and provide evidence that HTT influences the abundance and interaction landscape of proteins central to CAG repeat instability.

neuroscience↗

bioRxiv2026

Mismatch repair dissection by in vivo RNAi reveals dose-dependent modulators of somatic instability and proteome remodeling in Huntingtons disease

Human and mouse genetics have established mismatch repair (MMR) as a central mediator of somatic repeat expansion, a key pathogenic process in Huntingtons disease (HD) and related disorders. How individual MMR components function within the intact mammalian brain and interact with broader cellular networks remains poorly understood. We screened more than 500 chemically stabilized siRNAs targeting 10 MMR genes and used interventional RNAi in the Q111 HD mouse model to systematically dissect MMR function in vivo. MSH3 and PMS1 emerged as the most dose-sensitive regulators of somatic expansion but displayed markedly different effects on proteome stability. Quantitative proteomics generated an in vivo atlas of MMR component abundance and cross-regulation in the mammalian CNS, uncovering extensive connectivity between DNA repair, transcriptional regulation, chromatin remodeling, and mitochondrial biology. Together, these findings establish a systems-level framework linking MMR biology to neuronal function and offer mechanistic insight into selective neuronal vulnerability in HD.

neuroscience↗

bioRxiv2025

Lineage memory shapes viral resistance barriers in human skin

Individual cells within a given population exhibit striking variability in viral susceptibility, but it remains unknown whether this heterogeneity reflects memories encoded into the cellular lineage or true probabilistic variability. We used multi-color lineage tracing in a human primary organotypic skin model to reveal that viral resistance is encoded within specific cellular lineages. These lineages create distinct boundaries that block viral spread. Our lineage analyses in vitro confirmed that viral susceptibility exhibits strong heritability across cell generations, with siblings and cousins displaying remarkably similar infection outcomes. ATAC and proteomics profiling of resistant and susceptible clones revealed distinct epigenomic and proteomic states, with the transcription factor AP-1 emerging as a potential central regulator of lineage-encoded viral resistance. Inducing AP-1 activity with PMA rendered cells resistant to viral infection, suggesting a causative role in mediating resistance memory. Our findings demonstrate that antiviral resistance in human skin cells is encoded within cellular lineages and preserved through cell divisions, revealing how cell memory may shape infection dynamics and viral containment in tissues.

bioengineering↗

bioRxiv2024

Multi-epitope immunocapture of huntingtin reveals striatum-selective molecular signatures

Huntingtons disease (HD) is a debilitating neurodegenerative disorder affecting an individuals cognitive and motor abilities. HD is caused by mutation in the huntingtin gene producing a toxic polyglutamine-expanded protein (mHTT) and leading to degeneration in the striatum and cortex. Yet, the molecular signatures that underlie tissue-specific vulnerabilities remain unclear. Here, we investigate this aspect by leveraging multi-epitope protein interaction assays, subcellular fractionation, thermal proteome profiling, and genetic modifier assays. Use of human cell, mouse, and fly models afforded capture of distinct subcellular pools of epitope-enriched and tissue-dependent interactions linked to dysregulated cellular pathways and disease relevance. We established an HTT association with nearly all subunits of the transcriptional regulatory Mediator complex (20/26), with preferential enrichment of MED15 in the tail domain. Using HD and KO models, we find HTT modulates the subcellular localization and assembly of Mediator. We demonstrated striatal enriched and functional interactions with regulators of calcium homeostasis and chromatin remodeling, whose disease relevance was supported by HD fly genetic modifiers assays. Altogether, we offer insights into tissue- and localization-dependent (m)HTT functions and pathobiology.

neuroscience↗

bioRxiv2024

ARPE-19 Epithelial Cells Fail To Initiate Type-I Interferon Signaling in Response to Human Cytomegalovirus Infection.

ARPE-19 cells are a commonly used epithelial model for studying human cytomegalovirus (HCMV) infection. We recently found that ARPE-19 cells assume a mesenchymal phenotype when maintained at low confluency and that ARPE-19 cells resemble mesenchymal fibroblasts rather than epithelial cells in HCMV infection assays. Here, using comparative proteomics analysis, we find that subconfluent ARPE-19 cells are also deficient in their ability to initiate canonical type-I interferon signaling. Comparative proteomic analysis between subconfluent ARPE-19 and MRC-5 cells revealed a lack of canonical type-I interferon response in ARPE-19 cells upon HCMV infection, evidenced by the absence of interferon stimulated gene (ISG) induction. qRT-PCR and RNA-sequencing analysis revealed that ARPE-19 cells fail to initiate interferon-beta transcription in response to HCMV infection, yet they are competent to respond to exogenously interferon-b, indicating a failure in early pathogen detection. ARPE-19 cells showed low baseline levels of key intracellular pattern recognition receptors (PRRs) such as CGAS and IFI16, as well as the signaling molecule STING. This deficiency was associated with a failure to activate IRF3 phosphorylation, a crucial step in interferon signaling. These findings suggest an upstream defect in the early detection of viral components, likely due to reduced expression of critical PRRs. ARPE-19 cells may be inherently deficient in initiating interferon responses due to their derivation or possibly due to their origin from an immune-privileged tissue. Our results continue to highlight important phenotypic characteristics of the ARPE-19 cell line; important considerations for those using ARPE-19 cells as an experimental infection model for studying HCMV or other human viruses.

microbiology↗

bioRxiv2023

YlaN is an iron(II) binding protein that functions to relieve Fur-mediated repression of gene expression in Staphylococcus aureus

Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogen Staphylococcus aureus, the ferric uptake regulator (Fur) is the primary transcriptional regulator that controls the transcription of genes that code for Fe uptake and utilization proteins. YlaN was demonstrated to be essential in Bacillus subtilis unless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that YlaN is expendable in S. aureus; however, YlaN became essential upon Fe deprivation. A null fur allele bypassed the essentiality of YlaN. The transcriptional response of Fur derepression resulted in a reprogramming of metabolism to prioritize fermentative growth over respiratory growth. The absence of YlaN diminished the derepression of Fur-dependent transcription during Fe limitation. Bioinformatic analyses suggest that ylaN was recruited to Gram positive bacteria and once acquired was maintained in the genome as it co-evolved with Fur. Consistent with a role for YlaN in influencing Fur-dependent regulation, YlaN and Fur interacted in vivo. YlaN bound Fe(II) in vitro using oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe sensing and/or buffering. These findings have led to a model wherein YlaN is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction. ImportanceIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe can accumulate in the cytosol where it is toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent imbalance. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogen Staphylococcus aureus. Understanding the gene products involved in Fe ion regulation, uptake, and usage, as well as the physiological adaptations that S. aureus uses to survive in Fe-depleted conditions, will provide insight into the role that Fe has in pathogenesis. These data will also provide insight into the selective pressures imparted by the mammalian host.

microbiology↗

bioRxiv2022

Quantitative Proteomic Profiling of Murine Embryonic Heart Development Reveals a Role for the Mevalonate Pathway in Cardiomyocyte Proliferation

Defining the molecular mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the cardiac proteome at eight critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 identified proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways. Using this integrated dataset, we identified and established a functional role for the mevalonate pathway in the regulation of embryonic cardiomyocyte proliferation and cell signaling. Overall, our proteomic datasets are an invaluable resource for studying molecular events that regulate embryonic heart development and contribute to congenital heart disease.

developmental biology↗

bioRxiv2022

The interferon-inducible GTPase MxB promotes capsid disassembly and genome release of herpesviruses

Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid- host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.

microbiology↗

bioRxiv2020

Cardiac Sex Differences are Established Prior to Gonad Formation

Male and female disease states differ in their prevalence, treatment responses, and survival rates. In cardiac disease, women almost uniformly fare far worse than men. Though sex plays a critical role in cardiac disease, the mechanisms underlying sex differences in cardiac homeostasis and disease remain unexplained. Here, in adult and embryonic hearts we reveal sex-specific transcriptomes and proteomes and show that cardiac sex differences are predominately accounted for by post-transcriptional mechanisms. We found differential expression of male-female proteins in the cardiomyocytes. Using a quantitative proteomics-based approach, we characterized differential sex-specific enriched cardiac proteins, protein complexes, and biological sex processes in the context of global genetic diversity of the Collaborative Cross, an established surrogate for human diversity. We also found that sex differences in cardiac protein expression are established by both hormonal and sex chromosomal mechanisms. We have demonstrated the onset of sex-biased protein expression and discovered that sex disparities in heart tissue occur at the earliest stages of heart development at a period that preceeds mammalian gonadal development. Collectively, these findings may explain why congenital heart disease, a leading cause of death whose origin is often developmental, is sex biased. Our results reveal molecular foundations for differences in cardiac tissue that underlie sex disparities in health, disease, and treatment outcomes.

developmental biology↗

bioRxiv2018

Consensus clustering of herpesvirus protein interaction networks provides insights into their evolutionary relationship with bacteriophages

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the morbidity of some manifested symptoms but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterize herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, combining both experimentally supported and bioinformatic predictions. Our newly-developed consensus clustering approach allows us to extend the analysis beyond binary physical interactions and reveals higher order functional associations including that of pUS10 with capsid proteins. In-depth bioinformatics sequence analysis unraveled structural features of this 34-36 kDa protein reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are thought to share a common ancestry. This suggests that pUS10 could represent an evolutionary vestige between these two viral lineages. Using immunoaffinity purification-mass spectrometry we found that pUS10 specifically co-isolated with the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventual virion maturation. In summary, this study unveils new insights at both the system and molecular levels that can help better understand the complexity behind herpesvirus infections.

bioinformatics↗