Mass Spectrometry-Based Proteomics Reveals Distinct Mechanisms of Astrocyte Protein Secretion

The ability of astrocytes to secrete proteins subserves many of its known function, such as synapse formation during development and extracellular matrix remodeling after cellular injury. Protein secretion may also play an important, but less clear, role in the propagation of inflammatory responses and neurodegenerative disease pathogenesis. While potential astrocyte-secreted proteins may number in the thousands, known astrocyte-secreted proteins are less than 100. To address this fundamental deficiency, mass spectrometry-based proteomics and bioinformatic tools were utilized for global discovery, comparison, and quantification of astrocyte-secreted proteins. A primary mouse astrocyte cell culture model was used to generate a collection of astrocyte-secreted proteins termed the astrocyte secretome. A multidimensional protein and peptide separation approach paired with mass spectrometric analysis interrogated the astrocyte secretome under control and cytokine-exposed conditions, identifying cytokine-induced secreted proteins, while extending the depth of known astrocyte-secreted proteins to 169. Several of these proteins were likely secreted by non-conventional mechanisms. These non-conventional mechanisms were explored further using stable isotope labeling by amino acids in cell culture, revealing 12 putative non-conventionally secreted proteins. These qualitative and quantitative mass spectrometry approaches are broadly applicable for the study of cellular secretomes as well as for extension to in vivo secretomes

Systematic discovery of structural elements governing stability of mammalian messenger RNAs.

Decoding post-transcriptional regulatory programs in RNA is a critical step towards the larger goal of developing predictive dynamical models of cellular behaviour. Despite recent efforts, the vast landscape of RNA regulatory elements remains largely uncharacterized. A long-standing obstacle is the contribution of local RNA secondary structure to the definition of interaction partners in a variety of regulatory contexts, including--but not limited to--transcript stability, alternative splicing and localization. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (for example, human cardiac troponin T) or affects other aspects of RNA biology. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence. Here we present a computational framework based on context-free grammars and mutual information that systematically explores the immense space of small structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behaviour. By applying this framework to genome-wide human mRNA stability data, we reveal eight highly significant elements with substantial structural information, for the strongest of which we show a major role in global mRNA regulation. Through biochemistry, mass spectrometry and in vivo binding studies, we identified human HNRPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1, also known as HNRNPA2B1) as the key regulator that binds this element and stabilizes a large number of its target genes. We created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach could also be used to reveal the structural elements that modulate other aspects of RNA behaviour

YfmK is a Novel Nε-lysine Acetyltransferase that Directly Acetylates the Histone-like Protein HBsu in Bacillus Subtilis

Recently, Ne-lysine acetylation was realized to be a prevalent bacterial post-translational modification (PTM), contrary to the historical notion that this was a rare occurrence. Acetylation can impact protein function in multiple ways, by modifying conformation, interactions, subcellular localization or activity. In bacteria, hundreds of proteins are known to be acetylated, including those involved essential processes such as DNA replication, nucleoid organization, translation, cell shape, central carbon metabolism, and even several virulence factors. Despite the growing recognition that numerous proteins are acetylated, the biological significance of the vast majority of these modifications in any bacteria remains largely unknown. Previously, we characterized the Bacillus subtilis acetylome and found that the essential histone-like protein HBsu contains seven novel acetylation sites in vivo. HBsu is a bacterial nucleoid-associated protein, which is largely responsible for chromosome compaction and the coordination of DNA processes. Despite the lack of sequence or structural homology, it is generally considered to be a functional homolog of eukaryotic histones. We investigated whether acetylation is a regulatory component of the function of HBsu in nucleoid compaction. Using mutations that mimic the acetylated and unacetylated forms of the protein, we showed that the inability to acetylate key HBsu lysine residues results in a more compacted nucleoid. We further investigated the mechanism of HBsu acetylation. By screening knockouts of the ~50 putative Gcn5-N-acetyltransferase (GNAT)-domain encoding genes in B. subtilis for their effects on DNA compaction, five candidates were identified that may encode transacetylases acting on HBsu. Genetic bypass experiments demonstrated that two of these, YfmK and YdgE, can acetylate HBsu and their potential sites of action on HBsu were identified. Additionally, purified YfmK was able to directly acetylate HBsu in vitro,suggesting that it is the second identified protein acetyltransferase in B. subtilis. We propose that at least one physiological function of the acetylation of HBsu at key lysine residues is to regulate nucleoid compaction, analogously to the role of histone acetylation in eukaryotes. With the alarming rise in antibiotic resistance, the need to develop novel therapeutics is critical. Bacterial protein acetylation represents a world of untapped potential that may uncover new drug targets to replace or supplement our antiquated antibiotic arsenal. With proper study, the enzymes involved in regulation (i.e. acetylases and deacetylases) or the acetylated form of a key protein (i.e. virulence factors, essential genes, etc.) may provide valuable, druggable targets. The targeting of bacterial protein acetylation is a practical option, as targeting enzymes involved in acetylation regulation has been successful in treatment of certain cancers, latent viral and fungal infections

Measurement of the Mass and Stellar Population Distribution in M82 with the LBT

We present a K-band spectroscopic study of the stellar and gas kinematics, mass distribution, and stellar populations of the archetypical starburst galaxy M82. Our results are based on a single spectrum at a position angle of 67.5 degrees through the K-band nucleus. We used the CO stellar absorption band head at 2.29 {\mu}m (CO_2.29) to measure the rotation curve out to nearly 4 kpc radius on both the eastern and western sides of the galaxy. Our data show that the rotation curve is flat from 1 - 4 kpc. This stands in sharp contrast to some previous studies, which have interpreted H I and CO emission-line position-velocity diagrams as evidence for a declining rotation curve. The kinematics of the Br\gamma, H_2, and He I emission lines are consistent with, although characterized by slightly higher velocities than, the stellar kinematics. We derived M82's mass distribution from our stellar kinematic measurements and estimate its total dynamical mass is ~10^10 Msun. We measured the equivalent width of CO_2.29 (W_2.29) as a function of distance from the center of the galaxy to investigate the spatial extent of the red supergiant (RSG) population. The variation in W_2.29 with radius clearly shows that RSGs dominate the light inside 500 pc radius. M82's superwind is likely launched from this region, where we estimate the enclosed mass is <= 2 x 10^9 Msun.Comment: 6 pages, 6 figures. Submitted to ApJ. For a brief video explaining the key result of this paper, see http://www.youtube.com/user/OSUAstronom

The functional interactome landscape of the human histone deacetylase family

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102187/1/msb201326-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102187/2/msb201326.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102187/3/msb201326.reviewer_comments.pd

Formation of a TBX20-CASZ1 protein complex is protective against dilated cardiomyopathy and critical for cardiac homeostasis

By the age of 40, one in five adults without symptoms of cardiovascular disease are at risk for developing congestive heart failure. Within this population, dilated cardiomyopathy (DCM) remains one of the leading causes of disease and death, with nearly half of cases genetically determined. Though genetic and high throughput sequencing-based approaches have identified sporadic and inherited mutations in a multitude of genes implicated in cardiomyopathy, how combinations of asymptomatic mutations lead to cardiac failure remains a mystery. Since a number of studies have implicated mutations of the transcription factor TBX20 in congenital heart diseases, we investigated the underlying mechanisms, using an unbiased systems-based screen to identify novel, cardiac-specific binding partners. We demonstrated that TBX20 physically and genetically interacts with the essential transcription factor CASZ1. This interaction is required for survival, as mice heterozygous for both Tbx20 and Casz1 die post-natally as a result of DCM. A Tbx20 mutation associated with human familial DCM sterically interferes with the TBX20-CASZ1 interaction and provides a physical basis for how this human mutation disrupts normal cardiac function. Finally, we employed quantitative proteomic analyses to define the molecular pathways mis-regulated upon disruption of this novel complex. Collectively, our proteomic, biochemical, genetic, and structural studies suggest that the physical interaction between TBX20 and CASZ1 is required for cardiac homeostasis, and further, that reduction or loss of this critical interaction leads to DCM. This work provides strong evidence that DCM can be inherited through a digenic mechanism

Immunoglobulins Against Tyrosine-Nitrated Epitopes in Coronary Artery Disease

Background—Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine-nitrated proteins, a footprint of oxygen- and nitrogen-derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of patients with coronary artery disease (CAD). However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Methods and Results—Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of patients with CAD. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in patients with CAD (n=374) and subjects without CAD (non-CAD controls, n=313). A 10-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in patients with CAD (3.75±1.8 μg antibody Eq/mL plasma versus 0.36±0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. Conclusions—The results of this cross-sectional study suggest that posttranslational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neo-epitopes for the elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD

The Cardiac TBX5 Interactome Reveals a Chromatin Remodeling Network Essential for Cardiac Septation

Human mutations in the cardiac transcription factor gene TBX5 cause Congenital Heart Disease (CHD), however the underlying mechanism is unknown. We report characterization of the endogenous TBX5 cardiac interactome and demonstrate that TBX5, long considered a transcriptional activator, interacts biochemically and genetically with the Nucleosome Remodeling and Deacetylase (NuRD) repressor complex. Incompatible gene programs are repressed by TBX5 in the developing heart. CHD missense mutations that disrupt the TBX5-NuRD interaction cause depression of a subset of repressed genes. Furthermore, the TBX5-NuRD interaction is required for heart development. Phylogenetic analysis showed that the TBX5-NuRD interaction domain evolved during early diversification of vertebrates, simultaneous with the evolution of cardiac septation. Collectively, this work defines a TBX5-NuRD interaction essential to cardiac development and the evolution of the mammalian heart, and when altered may contribute to human CHD

A Gro/TLE-NuRD Corepressor Complex Facilitates Tbx20-Dependent Transcriptional Repression

The cardiac transcription factor Tbx20 has a critical role in the proper morphogenetic development of the vertebrate heart, and its misregulation has been implicated in human congenital heart disease. Although it is established that Tbx20 exerts its function in the embryonic heart through positive and negative regulation of distinct gene programs, it is unclear how Tbx20 mediates proper transcriptional regulation of its target genes. Here, using a combinatorial proteomic and bioinformatic approach, we present the first characterization of Tbx20 transcriptional protein complexes. We have systematically investigated Tbx20 protein-protein interactions by immunoaffinity purification of tagged Tbx20 followed by proteomic analysis using GeLC-MS/MS, gene ontology classification, and functional network analysis. We demonstrate that Tbx20 is associated with a chromatin remodeling network composed of TLE/Groucho co-repressors, members of the Nucleosome Remodeling and Deacetylase (NuRD) complex, the chromatin remodeling ATPases RUVBL1/RUVBL2, and the T-box repressor Tbx18. We determined that the interaction with TLE co-repressors is mediated via an eh1 binding motif in Tbx20. Moreover, we demonstrated that ablation of this motif results in a failure to properly assemble the repression network and disrupts Tbx20 function in vivo. Importantly, we validated Tbx20-TLE interactions in the mouse embryonic heart, and identified developmental genes regulated by Tbx20:TLE binding, thereby confirming a primary role for a Tbx20-TLE repressor complex in embryonic heart development. Together, these studies suggest a model in which Tbx20 associates with a Gro/TLE-NuRD repressor complex to prevent inappropriate gene activation within the forming heart