Biology subjects

Greene, G.

Publications and source records attributed to Greene, G..

4 recordsLinked to original sources

bioRxiv2026

Hierarchical cysteine oxidation controls reversible amyloid formation in an ankyrin repeat protein

The formation of amyloids, including functional amyloids, is observed for an increasing number of proteins but the molecular mechanisms that control this structural transition remain poorly understood. Here we report that the kinase inhibitor protein P18 (drP18) from Danio rerio (zebrafish), which contains two cysteine residues, undergoes a complex and hierarchical redox switch that strictly governs reversible amyloid formation. We identify cysteine 50 (C50) acting as a regulatory residue. Upon oxidation, C50 forms an intramolecular disulfide bond with the executioner cysteine 128 (C128), thereby blocking it. C50 can become S-glutathionylated, and upon oxidation, C128 then forms intermolecular disulfides that lead to rapid transition into amyloid fibrils. S-glutathionylation of C50 therefore enables amyloid formation of drP18 and the outcome is oxidant-dependent with diamide, hydrogen peroxide, peroxymonocarbonate and hypothiocyanous acid each leading to amyloid assembly with distinct kinetics and morphologies. These amyloids are fully reversible, where disulfide reduction is leading to disassembly. Whereas monomeric drP18 inhibits CDK4-mediated retinoblastoma phosphorylation, the amyloid conformation abolishes this inhibition, and reduction restores both structure and function. Expression of drP18 in zebrafish embryos yields Congo red-positive, oxidation-dependent aggregates in vivo. Together, our findings show that a regulatory cysteine controls an executioner cysteine to induce reversible, functional amyloid formation, revealing that proteins can encode sophisticated mechanisms to control amyloid assembly.

biochemistry↗

bioRxiv2024

A Boolean network model of hypoxia, mechanosensing and TGF-β signaling captures the role of phenotypic plasticity and mutations in tumor metastasis

The tumor microenvironment aids cancer progression by promoting several cancer hallmarks, independent of cancer-related mutations. Biophysical properties of this environment, such as the stiffness of the matrix cells adhere to and local cell density, impact proliferation, apoptosis, and the epithelial to mesenchymal transition (EMT). The latter is rate-limiting step for invasion and metastasis, enhanced in hypoxic tumor environments but hindered by soft matrices and/or high cell densities. As these influences are often studied in isolation, the crosstalk between hypoxia, biomechanical signals, and the classic EMT driver TGF-{beta} is not well mapped, limiting our ability to predict and anticipate cancer cell behaviors in changing tumor environments. To address this, we built a Boolean regulatory network model that integrates hypoxic signaling with a mechanosensitive model of EMT, which includes the EMT-promoting crosstalk of mitogens and biomechanical signals, cell cycle control, and apoptosis. Our model reproduces the requirement of Hif-1 for proliferation, the anti-proliferative effects of strong Hif-1 stabilization during hypoxia, hypoxic protection from anoikis, and hypoxia-driven mechanosensitive EMT. We offer experimentally testable predictions about the effect of VHL loss on cancer hallmarks, with or without secondary oncogene activation. Taken together, our model serves as a predictive framework to synthesize the signaling responses associated with tumor progression and metastasis in healthy vs. mutant cells. Our single-cell model is a key step towards more extensive regulatory network models that cover damage-response and senescence, integrating most cell-autonomous cancer hallmarks into a single model that can, in turn, control the behavior of in silico cells within a tissue model of epithelial homeostasis and carcinoma. Author SummaryThe cellular environment in and around a tumor can aid cancer progression by promoting several cancer hallmarks. This environment can affect growth and cell death, as well as a phenotype change that renders cells migratory and invasive: the epithelial to mesenchymal transition. Hypoxia (low oxygen availability) is known to promote this transition, while the attachment of cells to soft matrices or high cell density environments hinders it. These influences are often studied in isolation. As a result, their crosstalk is poorly understood. To address this, we have built a network model of cellular regulation that integrates a cells responses to hypoxia, the biophysical environment, and growth signals to model cell division, death, and the epithelial to mesenchymal transition in environments cells encounter during metastatic tumor progression. Our model reproduces a wide range of experimental cell responses and offers experimentally testable predictions about the emergence of cancer hallmarks driven mutations that affect the hypoxic response. Our single-cell model is a key step towards more extensive cell-scale models that also include cell aging and damage response. These, in turn, can serve as building blocks of a larger tissue model of healthy vs. cancerous epithelia.

cancer biology↗

bioRxiv2024

Age Dependent Integration of Cortical Progenitors Transplanted at the Adult CSF-Brain Interface

There has been renewed interest in neural transplantation of cells and tissues for brain repair. Recent studies have demonstrated the ability of transplanted neural precursor cells and in vitro grown organoids to mature and locally integrate into host brain neural circuitry. Much effort has focused on how the transplant behaves and functions after the procedure, but the extent to which the host brain can properly innervate the transplant, particularly in the context of aging, is largely unexplored. Here we report that transplantation of rat embryonic cortical precursor cells into the cerebrospinal fluid-subventricular zone (CSF-SVZ) of adult rat brains generates a brain-like tissue (BLT) at an ectopic site. This model allows for the assessment of long-range connectivity and cellular interactions between the transplant and the host brain as a function of host age. The transplanted precursor cells initially proliferate, then differentiate, and develop into mature BLTs, which receive supportive cellular components from the host including blood vessels, microglia, astrocytes, and oligodendrocytes. There was integration of the BLT into the host brain which occurred at all ages studied, suggesting that host age does not affect the maturation and integration of the transplant-derived BLT. Long-range axonal projections from the BLT into the host brain were robust throughout the different aged recipients. However, long-distance innervation originating from the host brain into the BLT significantly declined with age. This work demonstrates the feasibility and utility of integrating new neural tissue structures at ectopic sites into adult brain circuits to study host-transplant interactions.

neuroscience↗

bioRxiv2023

Structural determinants of pure antiestrogenicity

Inhibitory activities of antiestrogens on estrogen receptor alpha (ER) range from mixed antagonism/agonism (selective ER modulators; SERMs) to complete antiestrogenicity associated with accelerated ER turnover (selective ER degraders; SERDs). Here, we show using a panel of SERMs, SERDs and a PROTAC that efficient induction of ER SUMOylation better accounts for complete transcriptional repression than increased suppression of coactivator recruitment or accelerated ER degradation. Antiestrogen-induced ER SUMOylation depends on the hydrophobicity of N-terminal residues of ligand binding domain (LBD) helix 12 (H12). L536 mutations, including those occurring in endocrine therapy-resistant breast cancer, abolished SUMOylation with all antiestrogens. Structures of the L536S human ER LBD bound to fulvestrant analogs and molecular dynamics simulations predict dynamic side chain interactions with wild-type ER H12 in the coactivator-binding groove and model the impact of L536 mutations. Together, our results show that induced SUMOylation contributes to the complete suppression of ER activity and provide insights into its structural determinants.

molecular biology↗