64 research outputs found
N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation
© 2018 National Academy of Sciences. All Rights Reserved. The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington’s disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA ad-ducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems
Defining the proteolytic landscape during enterovirus infection.
Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease
CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases
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Chemical genetic approaches to study protein kinase signaling pathways
Protein kinase signaling is fundamental to regulating cellular homeostasis in the in every aspect of life. Protein kinases act by placing a phosphate group on a very specific subset of cellular proteins. The addition of a negative charged group on the surface of a protein can dramatically affect the folding, subcellular localization and activity of substrate proteins. Understanding the kinase responsible for a specific phosphorylation event has proven difficult as there are more than 500 mammalian kinases and greater than 50,000 phospho sites. The Shokat lab has pioneered a technique in which a particular kinase is engineered to accept either a specific inhibitor or modified substrate, which is normally not used by any other kinase. In this thesis we demonstrate the utility of this technique, by demonstrating its application to two important kinases. One of these kinases, NDR, is shown to be important in normal brain development, and we identify novel substrates that act directly downstream of NDR. Additionally, we identify novel targets of the kinase Aurora B, a kinase that is critical to normal chromosome segregation during cell division.In addition to these well-behaved kinases, we investigate a kinase, PINK1,that proves an exception to normal kinases. We show that PINK1 has the ability to utilize a kinetin triphosphate (KTP) catalytically better than its endogenous substrate, which we therefore call a neo-substrate. The development of a neo€-substrate or new substrate for the kinase PINK1 enhances the cellular activity of the disease-associated allele of PINK1 (G309D), effectively reversing the deleterious effect of the mutation. Since PINK1 is a key element in protection against mitochondrial damage and is genetically linked to Parkinson's Disease, our approach also has immediate therapeutic implications. Typically, overactive kinases such as oncogenic kinases provide the only opportunity for drug development because inhibitors of kinases are the only modality available for regulation of kinases. Therefore we believe this may represent a novel modality to regulate the activity of kinases
Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen
TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPase-activating protein (GAP) enzymes, and is exclusive to membrane-localized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection
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A Ras-like domain in the light intermediate chain bridges the dynein motor to a cargo-binding region.
Cytoplasmic dynein, a microtubule-based motor protein, transports many intracellular cargos by means of its light intermediate chain (LIC). In this study, we have determined the crystal structure of the conserved LIC domain, which binds the motor heavy chain, from a thermophilic fungus. We show that the LIC has a Ras-like fold with insertions that distinguish it from Ras and other previously described G proteins. Despite having a G protein fold, the fungal LIC has lost its ability to bind nucleotide, while the human LIC1 binds GDP preferentially over GTP. We show that the LIC G domain binds the dynein heavy chain using a conserved patch of aromatic residues, whereas the less conserved C-terminal domain binds several Rab effectors involved in membrane transport. These studies provide the first structural information and insight into the evolutionary origin of the LIC as well as revealing how this critical subunit connects the dynein motor to cargo
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