Biology subjects

Green, A. M.

Publications and source records attributed to Green, A. M..

6 recordsLinked to original sources

bioRxiv2024

ATR safeguards replication forks against APOBEC3B-induced toxic PARP1 trapping

ATR is the master safeguard of genomic integrity during DNA replication. Acute inhibition of ATR with ATR inhibitor (ATRi) triggers a surge in origin firing, leading to increased levels of single-stranded DNA (ssDNA) that rapidly deplete all available RPA. This leaves ssDNA unprotected and susceptible to breakage, a phenomenon known as replication catastrophe. However, the mechanism by which unprotected ssDNA breaks remains unclear. Here, we reveal that APOBEC3B is the key enzyme targeting unprotected ssDNA at replication forks, triggering a reaction cascade that induces fork collapse and PARP1 hyperactivation. Mechanistically, we demonstrate that uracils generated by APOBEC3B at replication forks are removed by UNG2, creating abasic sites that are subsequently cleaved by APE1 endonuclease. Moreover, we demonstrate that APE1-mediated DNA cleavage is the critical enzymatic step for PARP1 trapping and hyperactivation in cells, regardless of how abasic sites are generated on DNA. Finally, we show that APOBEC3B-induced toxic PARP1 trapping in response to ATRi drives cell sensitivity to ATR inhibition, creating to a context of synthetic lethality when combined with PARP inhibitors. Together, these findings reveal the mechanisms that cause replication forks to break during replication catastrophe and explain why ATRi-treated cells are particularly sensitive to PARP inhibitors.

molecular biology↗

bioRxiv2024

APOBEC3A drives metastasis of ovarian cancer by altering epithelial-to-mesenchymal transition

High-grade serous ovarian cancer (HGSOC) is the most prevalent and aggressive histological subtype of ovarian cancer, and often presents with metastatic disease. The drivers of metastasis in HGSOC remain enigmatic. APOBEC3A (A3A), an enzyme that generates mutations across various cancers, has been proposed as a mediator of tumor heterogeneity and disease progression. However, the role of A3A in HGSOC has not been explored. Through analysis of genome sequencing from primary HGSOC, we observed an association between high levels of APOBEC3 mutagenesis and poor overall survival. We experimentally addressed this correlation by modeling A3A activity in HGSOC cell lines and mouse models which resulted in increased metastatic behavior of HGSOC cells in culture and distant metastatic spread in vivo. A3A activity in both primary and cultured HGSOC cells yielded consistent alterations in expression of epithelial-mesenchymal-transition (EMT) genes resulting in hybrid EMT and mesenchymal signatures, and providing a mechanism for their increased metastatic potential. Our findings define the prevalence of A3A mutagenesis in HGSOC and implicate A3A as a driver of HGSOC metastasis via EMT, underscoring its clinical relevance as a potential prognostic biomarker. Our study lays the groundwork for the development of targeted therapies aimed at mitigating the deleterious impact of A3A-driven EMT in HGSOC.

cancer biology↗

bioRxiv2023

The SMC5/6 complex is required for maintenance of genome integrity upon APOBEC3A-mediated replication stress

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

molecular biology↗

bioRxiv2023

Reference frames for encoding of translation and tilt in the caudal cerebellar vermis

Many daily behaviors rely critically on estimates of our bodys motion and orientation in space. Vestibular signals are essential for such estimates but to contribute appropriately two key sets of computations are required. First, ambiguous motion information from the otolith organs must be combined with spatially transformed rotational signals (e.g., from the canals) to distinguish head translation from tilt. Second, tilt and translation estimates must be transformed from a head- to a body-centered reference frame to correctly interpret the bodys motion. Studies have shown that cells in the caudal cerebellar vermis (nodulus and ventral uvula, NU) reflect the output of the first set of computations to estimate translation and tilt. However, it remains unknown whether these estimates are encoded exclusively in head-centered coordinates or whether they reflect a further transformation towards body-centered coordinates. Here we addressed this question by examining how the 3D spatial tuning of otolith and canal signals on translation- and tilt-selective NU neurons varies with changes in head-re-body and body-re-gravity orientation. We show that NU cell tuning properties are consistent with head-centered coding of otolith signals during translation. Furthermore, while canals signals in the NU have been transformed into world-referenced estimates of reorientation relative to gravity (i.e., tilt), as needed to resolve the tilt-translation ambiguity, the resulting tilt estimates are encoded in head-centered coordinates. Our results thus suggest that body-centered motion and orientation estimates required for postural control, navigation and reaching are computed elsewhere either by further transforming NU outputs or via computations in other parallel pathways.

neuroscience↗

bioRxiv2023

Mucosal and systemic immune dynamics associated with COVID-19 outcomes: a longitudinal prospective clinical study

RationaleCOVID-19 severity varies widely; children and African Americans have low and high risk, respectively. Mechanistic data from these groups and the mucosa is lacking. ObjectivesTo quantify mucosal and systemic viral and immune dynamics in a diverse cohort to identify mechanisms underpinning COVID-19 severity and outcome predictors. MethodsIn this prospective study of unvaccinated children and adults COVID-19 outcome was based on an ordinal clinical severity scale. We quantified viral RNA, antigens, antibodies, and cytokines by PCR, ELISA, and Luminex from 579 longitudinally collected blood and nasal specimens from 78 subjects including 45 women and used modeling to determine functional relationships between these data. Measurements and Main ResultsCOVID-19 induced unique immune responses in African Americans (n=26) and children (n=20). Mild outcome was associated with more effective coordinated responses whereas moderate and severe outcomes had rapid seroconversion, significantly higher antigen, mucosal sCD40L, MCP-3, MCP-1, MIP-1, and MIP-1{beta}, and systemic IgA, IgM, IL-6, IL-8, IL-10, IL-15, IL-1RA, and IP-10, and uncoordinated early immune responses that went unresolved. Mucosal IL-8, IL-1{beta}, and IFN-{gamma} with systemic IL-1RA and IgA predicted COVID-19 outcomes. ConclusionsWe present novel mucosal data, biomarkers, and therapeutic targets from a diverse cohort. Based on our findings, children and African Americans with COVID-19 have significantly lower IL-6 and IL-17 levels which may reduce responsiveness to drugs targeting IL-6 and IL-17. Unregulated immune responses persisted indicating moderate to severe COVID-19 cases may require prolonged treatments. Reliance on slower acting adaptive responses may cause immune crisis for some adults who encounter a novel virus. At a Glance CommentaryO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSDespite the disparate outcomes for African Americans and children with COVID-19 and the vital role of mucosal immunity, the majority of mechanistic clinical studies lack these groups and mucosal assessments. To date, mucosal immune responses to SARS-CoV-2 has not been adequately described and we lack data from these understudied groups. What This Study Adds to the FieldThis was a prospective cohort study of children and adults with confirmed COVID-19. Mortality was low (2.5%). Severity outcomes were associated with African American Race, shortness of breath, fever, respiratory disease, high blood pressure, and diabetes. We systematically characterized viral and immune factors in the mucosa and periphery and observed that moderate and severe COVID-19 were associated with longer duration, impaired clearance, early overexuberant antibody and cytokine production that was sustained. This study demonstrates that African Americans are at high risk of severe COVID-19 and display unique mucosal and peripheral immune responses. Children with COVID-19 also had distinct immune responses. This illustrates the importance of vaccination and careful clinical oversight of these populations (e.g., lower IL-6 and IL-17 levels may diminish tocilizumab, siltuximab, secukinumab, and brodalumab efficacy). This study identified generalizable outcomes predictors, systemic IL-1RA with mucosal IL-1{beta} and IL-8, and demonstrated the utility of mucosal sampling from diverse cohorts.

immunology↗

bioRxiv2020

In Situ Optical Quantification of Extracellular Electron Transfer from Shewanella oneidensis using Plasmonic Metal Oxide Nanocrystals

Extracellular electron transfer (EET) is a critical form of microbial metabolism that enables respiration on a variety of inorganic substrates, including metal oxides. However, quantifying current generated by electroactive bacteria has been predominately limited to biofilms formed on electrodes. To address this, we developed a platform for quantifying EET flux from cell suspensions using aqueous dispersions of infrared plasmonic tin-doped indium oxide nanocrystals. Tracking the change in optical extinction during electron transfer enabled quantification of current generated by planktonic Shewanella oneidensis cultures. Using this method, we differentiated between starved and actively respiring cells, between cells of varying genotype, and between cells engineered to differentially express a key EET gene using an inducible genetic circuit. Overall, our results validate the utility of colloidally stable plasmonic metal oxide nanocrystals as quantitative biosensors in native biological environments and contribute to a fundamental understanding of planktonic S. oneidensis electrophysiology using simple in situ spectroscopy.

microbiology↗