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DOE OSTI2017

Deubiquitinase USP33 is negatively regulated by β-TrCP through ubiquitin-dependent proteolysis

Ubiquitin-mediated proteolysis regulates cellular levels of various proteins, and therefore plays important roles in controlling cell signaling and disease progression. The Skp1-Cul1-F-box ubiquitin ligase β-TrCP is recognized as an important negative regulator for numerous key signaling proteins. Recently, the deubiquitinases (DUBs) have turned out to be essential to regulate signaling pathways related to human diseases. However, whether β-TrCP is able to regulate the deubiquitinase family members remains largely unexplored. Here, we found that β-TrCP downregulated cellular levels of endogenous USP33. We also revealed that β-TrCP interacted with USP33 independently of the classic binding motif for β-TrCP, and mediated USP33 degradation via the ubiquitin proteasome pathway. Furthermore, we found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are required for the interaction between β-TrCP and USP33. Consequently, β-TrCP attenuated USP33-mediated inhibition of cell proliferation and cell invasion. Taken together, our study clarified that the E3 ligase β-TrCP regulates cellular USP33 levels by the ubiquitin-proteasomal proteolysis. - Highlights: •β-TrCP downregulates protein levels of USP33. •β-TrCP promotes USP33 ubiquitination and degradation. • The effect of β-TrCP on USP33 is independent of DSG motif. • β-TrCP attenuates USP33-mediated suppression of cell proliferation and invasion.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

DA-Raf, a dominant-negative antagonist of the Ras–ERK pathway, is a putative tumor suppressor

Highlights: • DA-Raf binds to oncogenic Ras and consequently suppresses the ERK pathway. • DA-Raf suppresses oncogenic Ras-induced transformation and tumorigenesis. • DA-Raf mutants cannot bind to oncogenic Ras or suppress the ERK pathway. • DA-Raf mutants cannot suppress oncogenic Ras-induced transformation and tumorigenesis. • DA-Raf is a tumor suppressor protein against Ras-induced tumorigenesis. Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf–MEK–ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras–ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

RalGPS2 is involved in tunneling nanotubes formation in 5637 bladder cancer cells

Highlights: • RalGPS2 is expressed in 5637 cancer cells and is essential for their growth. • RalGPS2 is required for TNT formation. • The overexpression of RalGPS2 PH domain markedly increased the number and length of TNT. • Interaction between RalA and Sec5 is required for TNT formation in 5637 cells. • RalGPS2 interacts with LST1 and RalA leading to TNT formation. RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Vitamin A deficiency induces endoplasmic reticulum stress and apoptosis in pancreatic islet cells: Implications of stearoyl-CoA desaturase 1-mediated oleic acid synthesis

Highlights: • For first time, here we report the possible underlying mechanism of vitamin A deficiency- induced pancreatic dysfunction. • Vitamin A deficiency altered the architecture and impaired the functions of pancreas. • Further, it increased the endoplasmic reticulum stress and reduced the SCD1-mediated monounsaturated fatty acid; oleic acid. • All these changes, possibly resulted in increased pancreatic islet cell death and thus decreased plasma insulin and glucagon levels. Previously, we reported that vitamin A deficiency resulted in the reduction of stearoyl-CoA desaturase 1 (SCD1) and monounsaturated fatty acid (MUFA) levels, which corroborated with attenuation of high fructose-induced hepatic steatosis. Here, we aimed at assessing the effect of vitamin A deficiency on SCD1, MUFA levels and their impact on pancreas’ structure and functions. Male weanling Wistar rats fed one of the four diets, namely control (Con), vitamin A-deficient (VAD), highfructose (HFr) and vitamin A-deficient diet with highfructose (VADHFr) for 16 weeks period. Compared to the control, feeding of VAD diet (alone or with HFr) resulted in pancreatic intra-islet vessel dilation and reduced plasma insulin, glucagon and C-peptide levels, however, glucose levels decreased only in VADHFr group. In line with plasma levels, VAD diet-fed animals displayed lower immunostaining for insulin and glucagon, which corroborated with increased apoptotic staining observed in the islet regions, possibly due to increased cellular stress, as indicated by high immunostaining for endothelial nitric oxide synthase (eNOS) and CCAAT/Enhancer-binding protein homologues protein (CHOP). On the other hand, it significantly decreased the SCD1 protein, which corroborated with reduced MUFA levels, particularly, oleic acid (C18:1), when compared to the control and HFr groups. In conclusion, chronic vitamin A deficiency altered the structure and functions of pancreas by diminishing the islet cells, possibly by inducing cellular stress-mediated apoptosis and decreasing SCD1-mediated oleic acid (C18:1) synthesis. Thus, the data suggest that unlike liver, the reduction in SCD1 and MUFA levels in the pancreas exerts deleterious effects on its functions and perturb the overall cellular metabolism.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux

Highlights: • Giant plasma membrane vesicles serve as a means to monitor cell surface cholesterol. • Increase in cholesterol efflux enhances giant plasma membrane vesicle formation. • Microtubule stabilization and acetylation promote cholesterol efflux in NPC disease. • Actin dynamics and Arl4c modulate lipid efflux and giant plasma membrane vesicles. Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease{sup 1–4}. Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Adaptation of metabolism to multicellular aggregation, hypoxia and obese stromal cell incorporation as potential measure of survival of ovarian metastases

Ovarian metastases exfoliate from the primary tumor and it is thought that aggregation supports their survival in the peritoneal cavity during dissemination but the underlying mechanisms are not clearly identified. We have previously shown that ovarian cancer cells acquire an increasingly glycolytic and metabolic flexible phenotype during progression. In the present study, we investigated how hypoxia, aggregation, and the incorporation of the obese stromal vascular fraction (SVF) affect cellular metabolism and the response to common anti-cancer and anti-diabetic drugs. Our results show a reduction of glucose uptake, lactate secretion, cellular respiration and ATP synthesis in response to hypoxia and aggregation, suggesting that the observed reduced proliferation of cells aggregated into spheroids is the result of a down-regulation of respiration. Recruitment of SVF to spheroids increased the spheroids invasive capacity but reduced respiration only in the most aggressive cells. Further, aggregation and hypoxia reduced the response to the metabolic drugs AICAR and metformin, and the chemotherapeutic agents cisplatin and paclitaxel. Our results suggest that the adaptation of cellular metabolism may contribute to enhanced survival under non-permissive conditions, and that these metabolic alterations may provide targets for future interventions that aim to enhance the survival of women with metastatic ovarian cancer.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Cyanine-Incorporated Amorphous Polymeric Carbon Dots for Optical Reactive Oxygen Species Sensing

Reactive oxygen species (ROS) are common cellular oxidants that when overproduced by cellular stressors cause harm to cells. Detection of ROS is of utmost importance to understanding a wide variety of cellular function and toxicity mechanisms. Conventional ROS fluorescence assays involve using a single dye to visualize the ROS quantity. Herein, we describe ROS-sensitive, fluorescent-dye-incorporated carbon dots with dual fluorescence capabilities and good biocompatibility. Carbon dots (CDs) made of citric acid and urea were synthesized with incorporated cyanine-3-amine (Cy3), a bright red fluorescent dye, to create Cy3-CDs. Additionally, to get Cy3 into the ROS-sensitive form, this work demonstrated that Cy3 alone and Cy3 within carbon dots can be electrochemically reduced to their colorless ROS-sensitive form. Cy3, CDs, and Cy3-CDs are all responsive to additions of superoxide, leading to an increase in the fluorescence. Overall, this work examines how O 2 •– and additional oxidizers interact with CDs, Cy3, and Cy3-CDs, and molecular-level hypotheses are explored that will inform the design of future carbon dot-based ROS sensors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

National Cancer Institute Think-Tank Meeting Report on Proteomic Cartography and Biomarkers at the Single-Cell Level: Interrogation of Premalignant Lesions

A Think-Tank Meeting was convened by the National Cancer Institute (NCI) to solicit experts’ opinion on the development and application of multi-omic single-cell analyses, and especially single-cell proteomics, to improve the development of a new generation of biomarkers for cancer risk, early detection, diagnosis, prognosis as well as to discuss the discovery of new targets for prevention and therapy. It is anticipated that such markers and targets will be based on cellular, subcellular, molecular and functional aberrations within the lesion and within individual cells. Additionally, single-cell proteomic data will be essential for the establishment of new tools that with searchable and scalable features that include spatial and temporal cartographies of premalignant and malignant lesions. Challenges and potential solutions that were discussed included: • The best way/s to analyze single-cells from fresh and preserved tissue • Detection and analysis of secreted molecules and from single cells, especially from a tissue slice • Detection of new, previously undocumented cell type/s in the premalignant and early stage cancer tissue microenvironment • Multi-omic integration of data to support and inform proteomic measurements • Subcellular organelles – identifying abnormal structure, function, distribution and location within individual premalignant and malignant cells • How to improve the dynamic range of single-cell proteomic measurements for discovery of differentially expressed proteins and their post translational modifications (PTM) • The depth of coverage measured concurrently using single-cell techniques • Quantitation - absolute or semiquantitative? • Single methodology or multiplexed combinations? • Application of analytical methods for identification of biologically significant subsets • Data visualization of N-dimensional datasets • How to construct intercellular signaling networks in individual cells within premalignant tumor microenvironments (TME) • Associations between intrinsic cellular processes and extrinsic stimuli • How to predict cellular responses to stress inducing stimuli • Identification of new markers for prediction of progression from precursor, benign and localized lesions to invasive cancer, based on spatial and temporal changes within individual cells • Identification of new targets for immunoprevention or immunotherapy – identification of neoantigens and surfactome of individual cells within a lesion

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Abnormal Cannabidiol Modulates Vitamin A Metabolism by Acting as a Competitive Inhibitor of CRBP1

Cellular retinol-binding proteins (CRBPs) facilitate the uptake and intracellular transport of vitamin A. They integrate retinoid metabolism, playing an important role in regulating the synthesis of bioactive vitamin A metabolites. Thus, CRBPs constitute potential pharmacological targets to modulate cellular retinoid status that in turn may have applications in the treatment of certain immunological, metabolic, and ocular disorders. Here in this paper we identify abnormal cannabidiol (abn-CBD) as a nonretinoid inhibitor of cellular retinol-binding protein 1 (CRBP1). X-ray crystal structures of CRBP1 in complex with abn-CBD and its derivatives revealed a distinctive mode of protein–ligand interaction and provided a molecular basis for the high affinity and selectivity of this compound. We demonstrated that abn-CBD modulates the flux of retinoids via the retinoid cycle in vivo. Furthermore, the biological activity of abn-CBD was evidenced by its ability to protect against light-induced retinal damage in Balb/cJ mice. Altogether, our findings indicate that targeting selected CRBPs with a small-molecule inhibitor can potentially lead to the development of new therapeutic agents to counteract diseases with etiologies involving imbalance in retinoid metabolism or signaling.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Novel Cell-Type-Specific Drought-Responsive Proteins in Root Tips of Field-Grown Perennial Switchgrass

The root-tip region of plants, including the root cap, forms the most basal terminal of the root and exhibits a high degree of cellular complexity in terms of morphology, cytological function, and interaction with environmental cues in the soil. Cells in this region follow a developmental trajectory, transitioning from stem cells to meristematic cells, and ultimately to fully differentiated cell types. However, our understanding of root-tip cell-type specific proteomic responses to abiotic stresses, such as drought, particularly under field conditions, remains limited. This study aimed to identify spatially resolved, cell type-specific proteomes in switchgrass (Panicum virgatum) root tips under drought stress. Root tips were collected from seven-year-old, field-grown switchgrass ‘Alamo’ plants excavated under both well-watered and long-term drought conditions. Cell type-specific proteins were identified using laser capture microdissection (LCM) coupled with nanoPOTS (Nanodroplet Processing in One Pot for Trace Samples) and nano-LC-MS proteomics analysis. Five distinct cell types were targeted: (1) cells in the quiescent center and stem cell niche (QuC), (2) protodermal epidermal cells (PEC) in the meristematic zone, (3) epidermal cells in the transition and elongation zones above the root cap (Epi), (4) peripheral root cap cells (PRC), forming 2–3 layers below the PEC and 1–2 layers above the root border cells, and (5) columella root cap cells (Col) comprising of the columella initials and a single underlying layer of cells undergoing active growth. Principal component analysis (PCA) revealed clear separation among the five targeted cell types, confirming distinct proteomic profiles. Proteins predominantly enriched in each cell type were linked to distinct cellular functions, with QuC cells showing involvement in chromosomal behavior, DNA replication, and mitosis—key processes for stem cell niche regulation. Drought stress resulted in alterations of proteostasis, as evidenced by significant decreases in ribosomal proteins and increases in protein synthesis inhibitors. Moreover, drought stress induced unique cell-type–specific proteins involved in phytohormone biosynthesis and signaling pathways, including auxin, cytokinin, and jasmonic acid. In particular, QuC cells were more highly enriched in proteins associated with DNA repair and mitotic processes. Metabolic pathways related to amino acids, carbohydrates, and lipids were differentially affected in a cell-type–dependent manner, whereas general stress-responsive proteins exhibited consistent changes across all five cell types. Overall, this study provides unique spatially resolved, cell-type-specific proteomic profiles in root tips, representing a significant advancement in our understanding of the cellular mechanisms underlying plant responses to drought stress in natural field conditions.

perennial grass↗

DOE OSTI2024

Emergence of Orchestrated and Dynamic Metabolism of Saccharomyces cerevisiae

Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. Furthermore, this study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Biodiversity and Stoichiometric Plasticity Increase Pico‐Phytoplankton Contributions to Marine Net Primary Productivity and the Biological Pump

Earth System Models generally predict increasing upper ocean stratification from 21st century warming, which will cause a decrease in the vertical nutrient flux forcing declines in marine net primary productivity (NPP) and carbon export. Recent advances in quantifying marine ecosystem carbon to nutrient stoichiometry have identified large latitudinal and biome variability, with low-latitude oligotrophic systems harboring pico-sized phytoplankton exhibiting large phosphorus to carbon cellular plasticity. The climate forced changes in nutrient flux stoichiometry and phytoplankton community composition are thus likely to alter the ocean's biogeochemical response and feedback with the carbon-climate system. We have added three pico-phytoplankton functional types within the Biogeochemical Elemental Cycling component of the Community Earth System Model while incorporating variable cellular phosphorus to carbon stoichiometry for all represented phytoplankton types. The model simulates Prochlorococcus and Synechococcus populations that dominate the productivity and sinking carbon export of the tropical and subtropical ocean, and pico-eukaryote populations that contribute significantly to productivity and export within the subtropical to mid-latitude transition zone, with the western subtropical regions of each basin supporting the most P-poor stoichiometries. Subtropical gyre recirculation regions along the poleward flanks of surface western boundary currents are identified as regional hotspots of enhanced carbon export exhibiting C-rich/P-poor stoichiometry, preferentially inhabited by pico-eukaryotes and diatoms. Collectively, pico-phytoplankton contribute ∼58% of global NPP and ∼46% of global particulate organic carbon export below 100 m through direct and ecosystem processing pathways. Biodiversity and cellular nutrient plasticity in marine pico-phytoplankton combine to increase their contributions to ocean productivity and the biological carbon pump.

Environmental Sciences & Ecology↗

DOE OSTI2017

Programming mRNA decay to modulate synthetic circuit resource allocation

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice

Cyclic GMP-AMP synthase is essential for innate immunity against infection and cellular damage, serving as a sensor of DNA from pathogens or mislocalized self-DNA. Upon binding double-stranded DNA, cyclic GMP-AMP synthase synthesizes a cyclic dinucleotide that initiates an inflammatory cellular response. Mouse studies that recapitulate causative mutations in the autoimmune disease Aicardi-Goutières syndrome demonstrate that ablating the cyclic GMP-AMP synthase gene abolishes the deleterious phenotype. Here, we report the discovery of a class of cyclic GMP-AMP synthase inhibitors identified by a high-throughput screen. These compounds possess defined structure-activity relationships and we present crystal structures of cyclic GMP-AMP synthase, double-stranded DNA, and inhibitors within the enzymatic active site. We find that a chemically improved member, RU.521, is active and selective in cellular assays of cyclic GMP-AMP synthase-mediated signaling and reduces constitutive expression of interferon in macrophages from a mouse model of Aicardi-Goutières syndrome. RU.521 will be useful toward understanding the biological roles of cyclic GMP-AMP synthase and can serve as a molecular scaffold for development of future autoimmune therapies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide’s affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Restructuring of Enterococcus faecalis biofilm architecture in response to antibiotic-induced stress

Bacterial biofilms are intrinsically resistant to antimicrobial treatment, which contributes to microbial persistence in clinical infections. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and is the most prevalent enterococcal species identified in healthcare-associated infections. Since intrinsic resistance to multiple antibiotics is common for enterococci, and antibiotic resistance is elevated in biofilm populations, it is imperative to understand the mechanisms involved. Previously, we identified two glycosyltransferase genes whose disruption resulted in impaired nascent biofilm formation in the presence of antibiotic concentrations subinhibitory for parent growth and biofilm formation. The glycosyltransferases are involved in synthesis of the cell-wall-associated rhamnopolysaccharide Epa. Here we examined the effect of epa mutations on the temporal development of E. faecalis biofilms, and on the effects of antibiotics on pre-formed biofilms using scanning electron microscopy. We show that ΔepaOX mutant cells arrange into complex multidimensional biofilms independent of antibiotic exposure, while parent cells form biofilms that are monolayers in the absence of antibiotics. Remarkably, upon exposure to antibiotics parent biofilm cells restructure into complex three-dimensional biofilms resembling those of the ΔepaOX mutant without antibiotics. All biofilms exhibiting complex cellular architectures were less structurally stable than monolayer biofilms, with the biofilm cells exhibiting increased detachment. Our results indicate that E. faecalis biofilms restructure in response to cellular stress whether induced by antibiotics in the case of parent cells, or by deficiencies in Epa composition for the ΔepaOX strain. The data demonstrate a link between cellular architecture and antibiotic resistance of E. faecalis biofilms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants

Abstract Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2019

Bright sub-20-nm cathodoluminescent nanoprobes for electron microscopy

Electron microscopy has been integral in our understanding of complex biological systems. Although electron microscopy reveals cellular morphology with nanoscale resolution, it does not provide information on the location of different types of proteins. An electron-microscopy-based bioimaging technology capable of localizing individual proteins and resolving protein-protein interactions with respect to cellular ultrastructure would provide important insights into the molecular biology of a cell. In this report, we synthesize small lanthanide-doped nanoparticles and measure the absolute photon emission rate of individual nanoparticles resulting from a given electron excitation flux (cathodoluminescence). Our results indicate that the optimization of nanoparticle composition, synthesis protocols and electron imaging conditions can lead to sub-20-nm nanolabels that would enable high signal-to-noise localization of individual biomolecules within a cellular context. In ensemble measurements, these labels exhibit narrow spectra of nine distinct colours, so the imaging of biomolecules in a multicolour electron microscopy modality may be possible.

77 NANOSCIENCE AND NANOTECHNOLOGY↗