Emma Monte

Conversion of proteins into biofuels by engineering nitrogen flux

Biofuels are currently produced from carbohydrates and lipids in feedstock. Proteins, in contrast, have not been used to synthesize fuels because of the difficulties of deaminating protein hydrolysates. Here we apply metabolic engineering to generate Escherichia coli that can deaminate protein hydrolysates, enabling the cells to convert proteins to C4 and C5 alcohols at 56% of the theoretical yield. We accomplish this by introducing three exogenous transamination and deamination cycles, which provide an irreversible metabolic force that drives deamination reactions to completion. We show that Saccharomyces cerevisiae, E. coli, Bacillus subtilis and microalgae can be used as protein sources, producing up to 4,035 mg/l of alcohols from biomass containing ∼22 g/l of amino acids. These results show the feasibility of using proteins for biorefineries, for which high-protein microalgae could be used as a feedstock with a possibility of maximizing algal growth and total CO2 fixation.

Neural Circuit-Specialized Astrocytes: Transcriptomic, Proteomic, Morphological, and Functional Evidence

Astrocytes are ubiquitous in the brain and are widely held to be largely identical. However, this view has not been fully tested, and the possibility that astrocytes are neural circuit specialized remains largely unexplored. Here, we used multiple integrated approaches, including RNA sequencing (RNA-seq), mass spectrometry, electrophysiology, immunohistochemistry, serial block-face-scanning electron microscopy, morphological reconstructions, pharmacogenetics, and diffusible dye, calcium, and glutamate imaging, to directly compare adult striatal and hippocampal astrocytes under identical conditions. We found significant differences in electrophysiological properties, Ca2+ signaling, morphology, and astrocyte-synapse proximity between striatal and hippocampal astrocytes. Unbiased evaluation of actively translated RNA and proteomic data confirmed significant astrocyte diversity between hippocampal and striatal circuits. We thus report core astrocyte properties, reveal evidence for specialized astrocytes within neural circuits, and provide new, integrated database resources and approaches to explore astrocyte diversity and function throughout the adult brain. VIDEO ABSTRACT.

High Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure

Background: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. Methods: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload–induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Results: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. Conclusions: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy.

Systems proteomics of cardiac chromatin identifies nucleolin as a regulator of growth and cellular plasticity in cardiomyocytes

Myocyte hypertrophy antecedent to heart failure involves changes in global gene expression, although the preceding mechanisms to coordinate DNA accessibility on a genomic scale are unknown. Chromatin-associated proteins alter chromatin structure by changing their association with DNA, thereby altering the gene expression profile. Little is known about the global changes in chromatin subproteomes that accompany heart failure, and the mechanisms by which these proteins alter chromatin structure. The present study tests the fundamental hypothesis that cardiac growth and plasticity in the setting of disease recapitulates conserved developmental chromatin remodeling events. We used quantitative proteomics to identify chromatin-associated proteins extracted via detergent and to quantify changes in their abundance during disease. Our study identified 321 proteins in this subproteome, demonstrating it to have modest conservation (37%) with that revealed using strong acid. Of these proteins, 176 exhibited altered expression during cardiac hypertrophy and failure; we conducted extensive functional characterization of one of these proteins, Nucleolin. Morpholino-based knockdown of nucleolin nearly abolished protein expression but surprisingly had little impact on gross morphological development. However, hearts of fish lacking Nucleolin displayed severe developmental impairment, abnormal chamber patterning and functional deficits, ostensibly due to defects in cardiac looping and myocyte differentiation. The mechanisms underlying these defects involve perturbed bone morphogenetic protein 4 expression, decreased rRNA transcription, and a shift to more heterochromatic chromatin. This study reports the quantitative analysis of a new chromatin subproteome in the normal and diseased mouse heart. Validation studies in the complementary model system of zebrafish examine the role of Nucleolin to orchestrate genomic reprogramming events shared between development and disease.

Epigenomes: The missing heritability in human cardiovascular disease?

Cardiovascular disease is a tremendous burden on human health and results from malfunction of various networks of biological molecules in the context of environmental stress. Despite strong evidence of heritability, many common forms of heart disease (heart failure in particular) have not yielded to genome‐wide association studies to identify causative mutations acting via the disruption of individual molecules. Increasing evidence suggests, however, that genetic variation in noncoding regions is strongly linked to disease susceptibility. We hypothesize that epigenomic variation may engender different chromatin environments in the absence of (or in parallel with) changes in protein or mRNA sequence and abundance. In this manner, distinct—genetically encoded—chromatin environments can exhibit distinct responses to environmental stresses that cause heart failure, explaining a significant portion of the altered susceptibility that is observed in human disease.

Structural considerations for chromatin state models with transcription as a functional readout

Lacking from the rapidly evolving field of chromatin regulation is a discrete model of chromatin states. We propose that each state in such a model should meet two conditions: a structural component and a quantifiable effect on transcription. The practical benefits to the field of a model with greater than two states (including one with six states, as described herein) would be to improve interpretation of data from disparate organ systems, to reflect temporal and developmental dynamics and to integrate the, at present, conceptually and experimentally disparate analyses of individual genetic loci (in vitro or using single gene approaches) and genome‐wide features (including ChlP‐seq, chromosomal capture and mRNA expression via microarrays/sequencing).

Relationship of disease-associated gene expression to cardiac phenotype is buffered by genetic diversity and chromatin regulation

Expression of a cohort of disease-associated genes, some of which are active in fetal myocardium, is considered a hallmark of transcriptional change in cardiac hypertrophy models. How this transcriptome remodeling is affected by the common genetic variation present in populations is unknown. We examined the role of genetics, as well as contributions of chromatin proteins, to regulate cardiac gene expression and heart failure susceptibility. We examined gene expression in 84 genetically distinct inbred strains of control and isoproterenol-treated mice, which exhibited varying degrees of disease. Unexpectedly, fetal gene expression was not correlated with hypertrophic phenotypes. Unbiased modeling identified 74 predictors of heart mass after isoproterenol-induced stress, but these predictors did not enrich for any cardiac pathways. However, expanded analysis of fetal genes and chromatin remodelers as groups correlated significantly with individual systemic phenotypes. Yet, cardiac transcription factors and genes shown by gain-/loss-of-function studies to contribute to hypertrophic signaling did not correlate with cardiac mass or function in disease. Because the relationship between gene expression and phenotype was strain specific, we examined genetic contribution to expression. Strikingly, strains with similar transcriptomes in the basal heart did not cluster together in the isoproterenol state, providing comprehensive evidence that there are different genetic contributors to physiological and pathological gene expression. Furthermore, the divergence in transcriptome similarity versus genetic similarity between strains is organ specific and genome-wide, suggesting chromatin is a critical buffer between genetics and gene expression.

Systems Proteomics of Healthy and Diseased Chromatin

Differences in chromatin-associated proteins allow the same genome to participate in multiple cell types and to respond to an array of stimuli in any given cell. To understand the fundamental properties of chromatin and to reveal its cell- and/or stimulus-specific behaviors, quantitative proteomics is an essential technology. This chapter details the methods for fractionation and quantitative mass spectrometric analysis of chromatin from hearts or isolated adult myocytes, detailing some of the considerations for applications to understanding heart disease. The state-of-the-art methodology for data interpretation and integration through bioinformatics is reviewed.

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,(1) the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.(2) Transcriptional regulation during development and disease have been well studied in this organ,(3-5) but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.(6) In the developed world, heart disease is the number one cause of mortality for both men and women.(7) Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options. Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,(8-13) until recently(14) there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.(15) In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.(16) Additionally, cardiomyocytes are 40% mitochondria by volume(17) which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Epigenomic Disruption of Cardiovascular Care: What It Will Take

Hypothesis-driven research, while indispensable for generating new knowledge, is often not the source of new clinical advances. Omics technologies can theoretically provide a more quantitative and precise understanding of human health. However, this requires revaluation of some of the central assumptions of basic research: that rodents faithfully model human physiology, that better understanding of biological mechanism is the fastest way to new therapies, and that patients are healthcare customers, rather than participants. Article, see p 1754 Coronary heart disease (CHD) is a significant cause of morbidity and mortality worldwide. The largest population on earth, combined with rapid industrialization in recent decades, make China’s CHD epidemic particularly large and dynamic. Estimates attribute 22% of cardiovascular deaths and 9% of total deaths to CHD in urban populations (13% and 4% for rural populations, respectively).1 In the United States, CHD accounts for 45% of cardiovascular and 14% of total deaths.2 What is the goal of precision health in CHD, a disease in which the risk factors are well known and largely modifiable? Unlike genetic markers, epigenetic markers have the potential to change behavior because they are themselves the result of environmental factors interacting with the genome. DNA methylation is a proven marker for and therapeutic target in cancer.3 Methylation arrays have enabled investigation of large cohorts across a range of common diseases.4 DNA methylation signatures lurking in blood have been shown to correlate with …