Anthony A. High

Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery.

TDP-43 is a highly conserved and ubiquitously expressed member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins. Recently, TDP-43 was shown to be a major disease protein in the ubiquitinated inclusions characteristic of most cases of amyotrophic lateral sclerosis (ALS), tau-negative frontotemporal lobar degeneration (FTLD), and inclusion body myopathy. In these diseases, TDP-43 is redistributed from its predominantly nuclear location to ubiquitin-positive, cytoplasmic foci. The extent to which TDP-43 drives pathophysiology is unknown, but the identification of mutations in TDP-43 in familial forms of ALS and FTLD-U suggests an important role for this protein in pathogenesis. Little is known about TDP-43 function and only a few TDP-43 interacting proteins have been previously identified, which makes further insight into both the normal and pathological functions of TDP-43 difficult. Here we show, via a global proteomic approach, that TDP-43 has extensive interaction with proteins that regulate RNA metabolism. Some interactions with TDP-43 were found to be dependent on RNA-binding, whereas other interactions are RNA-independent. Disease-causing mutations in TDP-43 (A315T and M337V) do not alter its interaction profile. TDP-43 interacting proteins largely cluster into two distinct interaction networks, a nuclear/splicing cluster and a cytoplasmic/translation cluster, strongly suggesting that TDP-43 has multiple roles in RNA metabolism and functions in both the nucleus and the cytoplasm. Finally, we found numerous TDP-43 interactors that are known components of stress granules, and indeed, we find that TDP-43 is also recruited to stress granules.

mTORC1 Signaling under Hypoxic Conditions Is Controlled by ATM-Dependent Phosphorylation of HIF-1α

The mTOR complex-1 (mTORC1) coordinates cell growth and metabolism, acting as a restriction point under stress conditions such as low oxygen tension (hypoxia). Hypoxia suppresses mTORC1 signaling. However, the signals by which hypoxia suppresses mTORC1 are only partially understood, and a direct link between hypoxia-driven physiological stress and the regulation of mTORC1 signaling is unknown. Here we show that hypoxia results in ataxia telangiectasia mutated (ATM)-dependent phosphorylation of hypoxia-inducible factor 1-alpha (HIF-1α) on serine(696) and mediates downregulation of mTORC1 signaling. Deregulation of these pathways in pediatric solid tumor xenografts suggests a link between mTORC1 dysregulation and solid tumor development and points to an important role for hypoxic regulation of mTORC1 activity in tumor development.

A20-binding inhibitor of NF-κB (ABIN1) controls Toll-like receptor-mediated CCAAT/enhancer-binding protein β activation and protects from inflammatory disease

Toll-like receptors (TLRs) are expressed on innate immune cells and trigger inflammation upon detection of pathogens and host tissue injury. TLR-mediated proinflammatory-signaling pathways are counteracted by partially characterized anti-inflammatory mechanisms that prevent exaggerated inflammation and host tissue damage as manifested in inflammatory diseases. We biochemically identified a component of TLR-signaling pathways, A20-binding inhibitor of NF-κB (ABIN1), which recently has been linked by genome-wide association studies to the inflammatory diseases systemic lupus erythematosus and psoriasis. We generated ABIN1-deficient mice to study the function of ABIN1 in vivo and during TLR activation. Here we show that ABIN1-deficient mice develop a progressive, lupus-like inflammatory disease characterized by expansion of myeloid cells, leukocyte infiltrations in different parenchymatous organs, activated T and B lymphocytes, elevated serum Ig levels, and the appearance of autoreactive antibodies. Kidneys develop glomerulonephritis and proteinuria, reflecting tissue injury. Surprisingly, ABIN1-deficient macrophages exhibit normal regulation of major proinflammatory signaling pathways and mediators but show selective deregulation of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) and its target genes, such as colony-stimulating factor 3 (Csf3), nitric oxide synthase, inducible (Nos2), and S100 calcium-binding protein A8 (S100a8). Their gene products, which are intimately linked to innate immune cell expansion (granulocyte colony-stimulating factor), cytotoxicity (inducible nitric oxide synthase), and host factor-derived inflammation (S100A8), may explain, at least in part, the inflammatory phenotype observed. Together, our data reveal ABIN1 as an essential anti-inflammatory component of TLR-signaling pathways that controls C/EBPβ activity.

Large-scale analysis of thermostable, mammalian proteins provides insights into the intrinsically disordered proteome

Intrinsically disordered proteins are predicted to be highly abundant and play broad biological roles in eukaryotic cells. In particular, by virtue of their structural malleability and propensity to interact with multiple binding partners, disordered proteins are thought to be specialized for roles in signaling and regulation. However, these concepts are based on in silico analyses of translated whole genome sequences, not on large-scale analyses of proteins expressed in living cells. Therefore, whether these concepts broadly apply to expressed proteins is currently unknown. Previous studies have shown that heat-treatment of cell extracts lead to partial enrichment of soluble, disordered proteins. On the basis of this observation, we sought to address the current dearth of knowledge about expressed, disordered proteins by performing a large-scale proteomics study of thermostable proteins isolated from mouse fibroblast cells. With the use of novel multidimensional chromatography methods and mass spectrometry, we identified a total of 1320 thermostable proteins from these cells. Further, we used a variety of bioinformatics methods to analyze the structural and biological properties of these proteins. Interestingly, more than 900 of these expressed proteins were predicted to be substantially disordered. These were divided into two categories, with 514 predicted to be predominantly disordered and 395 predicted to exhibit both disordered and ordered/folded features. In addition, 411 of the thermostable proteins were predicted to be folded. Despite the use of heat treatment (60 min at 98 degrees C) to partially enrich for disordered proteins, which might have been expected to select for small proteins, the sequences of these proteins exhibited a wide range of lengths (622 +/- 555 residues (average length +/- standard deviation) for disordered proteins and 569 +/- 598 residues for folded proteins). Computational structural analyses revealed several unexpected features of the thermostable proteins: (1) disordered domains and coiled-coil domains occurred together in a large number of disordered proteins, suggesting functional interplay between these domains; and (2) more than 170 proteins contained lengthy domains (>300 residues) known to be folded. Reference to Gene Ontology Consortium functional annotations revealed that, while disordered proteins play diverse biological roles in mouse fibroblasts, they do exhibit heightened involvement in several functional categories, including, cytoskeletal structure and cell movement, metabolic and biosynthetic processes, organelle structure, cell division, gene transcription, and ribonucleoprotein complexes. We believe that these results reflect the general properties of the mouse intrinsically disordered proteome (IDP-ome) although they also reflect the specialized physiology of fibroblast cells. Large-scale identification of expressed, thermostable proteins from other cell types in the future, grown under varied physiological conditions, will dramatically expand our understanding of the structural and biological properties of disordered eukaryotic proteins.

Novel Function of Prothymosin Alpha as a Potent Inhibitor of Human Immunodeficiency Virus Type 1 Gene Expression in Primary Macrophages

ABSTRACT CD8+ T lymphocytes control human immunodeficiency virus type 1 (HIV-1) infection by a cytotoxic major histocompatibility complex-restricted pathway as well as by secretion of noncytotoxic soluble inhibitory factors. Several components of CD8+ cell supernatants have been identified that contribute to the latter activity. In this study we report that prothymosin alpha (ProTα), a protein found in the cell culture medium of the herpesvirus saimiri-transformed CD8+ T-cell line, K#1 50K, has potent HIV-1-inhibitory activity. Depletion of native ProTα from an HIV-1-inhibitory fraction of CD8+ cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProTα is an abundant, acidic peptide that has been reported to be localized in the nucleus and associated with cell proliferation and activation of transcription. In this report we demonstrate that ProTα suppresses HIV-1 replication, its activity is target cell specific, and inhibition occurs following viral integration. Native and recombinant ProTα protein potently inhibit HIV-1 long terminal repeat (LTR)-driven gene expression in macrophages. Furthermore studies using different promoters in lentiviral vectors (cytomegalovirus and phosphoglycerate kinase) revealed that suppression of viral replication by ProTα is not HIV LTR specific.

Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor

ABSTRACT Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense.

The E3 Ubiquitin Ligase Mind Bomb-2 (MIB2) Protein Controls B-cell CLL/Lymphoma 10 (BCL10)-dependent NF-κB Activation

Background: BCL10 is an essential molecule for the activation of the transcription factor NF-κB in numerous immune receptor signaling pathways. Results: Identification of the E3 ubiquitin ligase MIB2 as part of the activated BCL10 complex controlling NF-κB activity. Conclusion: MIB2 mediates BCL10-dependent NF-κB activation. Significance: Identification of MIB2 as a regulator of important immune receptor signaling pathways. B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

Hematopoietic progenitor cell lines with myeloid and lymphoid potential

Investigation of immune-cell differentiation and function is limited by shortcomings of suitable and scalable experimental systems. Here we show that retroviral delivery of an estrogen-regulated form of Hoxb8 into mouse bone marrow cells can be used along with Flt3 ligand to conditionally immortalize early hematopoietic progenitor cells (Hoxb8-FL cells). Hoxb8-FL cells have lost self-renewal capacity and potential to differentiate into megakaryocytes and erythrocytes but retain the potential to differentiate into myeloid and lymphoid cells. They differentiate in vitro and in vivo into macrophages, granulocytes, dendritic cells, B lymphocytes and T lymphocytes that are phenotypically and functionally indistinguishable from their primary counterparts. Quantitative in vitro assays indicate that myeloid and B-cell potential of Hoxb8-FL cells is comparable to that of primary lymphoid-primed multipotent progenitors, whereas T-cell potential is diminished. The simplicity of this system and the unlimited proliferative capacity of Hoxb8-FL cells will enable studies of immune-cell differentiation and function.

Identification and characterization of phosphorylation sites within the pregnane X receptor protein

Pregnane X receptor (PXR) is a xenobiotic sensor regulating the expression of genes involved in xenobiotic detoxification and elimination. Phosphorylation plays an important role in modulating PXR activity and several phosphorylation sites have been predicted and characterized in in vitro experiments. Although PXR has been shown to be a phosphoprotein in vivo, the exact residues that are phosphorylated remain elusive. Using mass spectrometry, we identified for the first time S114, T133/135, S167, and S200 residues that are phosphorylated in PXR following an in vitro kinase assay using cyclin-dependent kinase 2. We further found that the phosphorylation at S114, T133, and T135 occurred in PXR isolated from cells. We tested the phosphodeficient and phosphomimetic mutants corresponding to all the sites identified and determined that phosphorylation at S114 attenuates the transcriptional activity of PXR, consistent with the observation that the S114D mutant displayed reduced association with the PXR-targeted DNA response element. Phosphomimetic mutations at either T133 or T135 did not show a significant change in transcriptional activity however, the dual phosphomimetic mutant T133D/T135D displayed reduced transcriptional activity. Subcellular localization studies showed a varied distribution of the mutants suggesting that the regulation of PXR is much more complex than what we can observe by just overexpressing the mutants. Thus, our results provide the first direct evidence that PXR is phosphorylated at specific residues and suggest that further investigation is warranted to fully understand the regulation of PXR by phosphorylation.

Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer's Disease

The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer’s disease (AD) the aggregation of the U1-70K splicing factor and abnormal RNA processing. Here, we present that U1-70K can be cleaved into an N-terminal truncation (N40K) in ∼50% of AD cases, and the N40K abundance is inversely proportional to the total level of U1-70K. To map the cleavage site, we compared tryptic peptides of N40K and stable isotope labeled U1-70K by liquid chromatography–tandem mass spectrometry (MS), revealing that the proteolysis site is located in a highly repetitive and hydrophilic domain of U1-70K. We then adapted Western blotting to map the cleavage site in two steps: (i) mass spectrometric analysis revealing that U1-70K and N40K share the same N-termini and contain no major modifications; (ii) matching N40K with a series of six recombinant U1-70K truncations to define the cleavage site within a small region (Arg300 ± 6 residues). Finally, N40K expression led to substantial degeneration of rat primary hippocampal neurons. In summary, we combined multiple approaches to identify the U1-70K proteolytic site and found that the N40K fragment might contribute to neuronal toxicity in Alzheimer’s disease.