Biology subjects

Hughes, M. P.

Publications and source records attributed to Hughes, M. P..

9 recordsLinked to original sources

bioRxiv2023

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

cell biology↗

bioRxiv2023

Dysregulated Wnt and NFAT signaling in a Parkinson's disease LRRK2 G2019S knock-in model

BackgroundParkinsons disease (PD) is a progressive late-onset neurodegenerative disease leading to physical and cognitive decline. Mutations of leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. LRRK2 is a complex scaffolding protein with known regulatory roles in multiple molecular pathways. Two prominent examples of LRRK2-modulated pathways are Wingless/Int (Wnt) and nuclear factor of activated T-cells (NFAT) signaling. Both are well described key regulators of immune and nervous system development as well as maturation. The aim of this study was to establish the physiological and pathogenic role of LRRK2 in Wnt and NFAT signaling in the brain, as well as the potential contribution of the non-canonical Wnt/Calcium pathway. MethodsIn vivo cerebral Wnt and NFATc1 signaling activity was quantified in LRRK2 G2019S mutant knock-in (KI) and LRRK2 knockout (KO) male and female mice with repeated measures over 28 weeks, employing lentiviral luciferase biosensors, and analyzed using a mixed-effect model. To establish spatial resolution, we investigated tissues, and primary neuronal cell cultures from different brain regions combining luciferase signaling activity, immunohistochemistry, qPCR and western blot assays. Results were analyzed by unpaired t-test with Welchs correction or 2-way ANOVA with post hoc corrections. ResultsIn vivo Wnt signaling activity in LRRK2 KO and LRRK2 G2019S KI mice was increased significantly [~]3-fold, with a more pronounced effect in males ([~]4-fold) than females ([~]2-fold). NFATc1 signaling was reduced [~]0.5-fold in LRRK2 G2019S KI mice. Brain tissue analysis showed region-specific expression changes in Wnt and NFAT signaling components. These effects were predominantly observed at the protein level in the striatum and cerebral cortex of LRRK2 KI mice. Primary neuronal cell culture analysis showed significant genotype-dependent alterations in Wnt and NFATc1 signaling under basal and stimulated conditions. Wnt and NFATc1 signaling was primarily dysregulated in cortical and hippocampal neurons respectively. ConclusionsOur study further built on knowledge of LRRK2 as a Wnt and NFAT signaling protein. We identified complex changes in neuronal models of LRRK2 PD, suggesting a role for mutant LRRK2 in the dysregulation of NFAT, and canonical and non-canonical Wnt signaling.

neuroscience↗

bioRxiv2022

CLN7 mutation causes aberrant redistribution of protein isoforms and contributes to Batten disease pathobiology

The variant late infantile form of the inherited neurodegenerative Batten disease (BD) is caused by mutations in the CLN7/MFSD8 gene and represents a strong candidate for gene therapy. Post-natal intracerebral administration of AAV9-hCLN7 to Cln7{Delta}ex2 knockout mice resulted in extended lifespan but dose escalation resulted in reduced acuity in neurophysiology tests, cerebral atrophy and elevated neuroinflammation. Comparing patient and control iPSC-derived neural progenitor cells (iNPC) we discovered that CLN7 localizes to the nucleus as well as the endolysosomal network and is differentially distributed in BD iNPC. Proteomics identified a profound nuclear defect in BD iNPC that compounds with mitochondrial and lysosomal metabolic defects resulting in elevated apoptosis. We further identified a 50kDa common nuclear CLN7 isoform and a 37kDa isoform that accumulates only in BD iNPC nuclei. Our findings suggest that successful treatment of CLN7 BD will require combinatorial therapies addressing both loss and aberrant gain of protein function.

cell biology↗

bioRxiv2022

A Spatiotemporal Reconstruction of the C. elegans Pharyngeal Cuticle Reveals a Structure Rich in Phase-Separating Proteins

Roughly 4.5 million species of ecdysozoan animals repeatedly shed their old cuticle and construct a new one underneath to accommodate growth. How cuticles are constructed is not well understood. Here, we systematically mine gene expression datasets to uncover the spatiotemporal blueprint for how the chitin-based pharyngeal cuticle of the nematode Caenorhabditis elegans is built. We demonstrate that the blueprint correctly predicts expression patterns and functional relevance to cuticle development. We find that as larvae prepare to molt, catabolic enzymes are upregulated and the genes that encode chitin synthase, chitin cross-linkers, and homologs of amyloid regulators subsequently peak in expression. 48% of the gene products secreted during the molt are predicted to be intrinsically disordered proteins (IDPs), many of which belong to four distinct families that are expressed in overlapping waves. These include the IDPAs, IDPBs, and IDPCs that are introduced for the first time here. We find that all four families have sequence properties known to drive phase separation and show in vitro phase separation for one of these proteins. This systematic analysis reveals the massive contribution that IDPs make to the cuticle and highlights how reversibly phase-separating materials may facilitate cuticle disassembly and reassembly during the molt.

systems biology↗

bioRxiv2020

The prevalence and distribution in genomes of low-complexity, amyloid-like, reversible, kinked segment (LARKS), a common structural motif in amyloid-like fibrils

Membraneless Organelles (MLOs) are vital and dynamic reaction centers in cells that organize metabolism in the absence of a membrane. Multivalent interactions between protein Low-Complexity Domains (LCDs) contribute to MLO organization. Our previous work used computational methods to identify structural motifs termed Low-complexity Amyloid-like Reversible Kinked Segments (LARKS) that can phase-transition to form hydrogels and are common in human proteins that participate in MLOs. Here we searched for LARKS in proteomes of six model organisms: Homo sapiens, Drosophila melanogaster, Plasmodium falciparum, Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Escherichia coli. We find LARKS are abundant in M. tuberculosis, D. melanogaster, and H. sapiens, but not in S. cerevisiae or P. falciparum. Abundant LARKS require high glycine content, which enables kinks to form in LARKS as is illustrated in the known LARKS-rich amyloid structures of TDP43, FUS, and hnRNPA2, three proteins that participate in MLOs. These results support the idea of LARKS as an evolved structural motif and we offer the LARKSdb webserver which permits users to search for LARKS in their protein sequences of interest.

biochemistry↗

bioRxiv2020

The cryoEM structure of the fibril-forming low-complexity domain of hnRNPA2 reveals distinct differences from pathogenic amyloid and shows how mutation converts it to the pathogenic form

hnRNPA2 is one of a group of human ribonucleoproteins (RNPs) involved in RNA metabolism which form fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and that missense mutations in this domain are found in the disease multisystem proteinopathy (MSP) with symptoms indistinguishable from ALS and FTD. However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into {beta}-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both their reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to pathogenic form.

molecular biology↗

bioRxiv2019

The α-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure

Aggregation of -synuclein is a defining molecular feature of Parkinsons disease, Lewy Body Dementia, and Multiple Systems Atrophy. Hereditary mutations in -synuclein are linked to both Parkinsons disease and Lewy Body Dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy Body Dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on -synuclein fibril structure is fundamental to -synuclein biology. We therefore determined the cryoEM structure of -synuclein fibrils containing the hereditary E46K mutation. The 2.5 [A] structure reveals a symmetric double protofilament in which the molecules adopt a vastly re-arranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which forms the E46-K80 salt-bridge in the wild-type fibril structure. We hypothesize that under our conditions the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts -synuclein into a kinetic trap - a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure. Significance StatementParkinsons is the second most prevalent neurodegenerative condition, leading to movement disorders, and dementia in some cases. Because of the strong association of this condition with amyloid aggregates of the protein -synuclein, structural understanding of these amyloid aggregates may be the path to eventual therapies. Our study of the structure of a variant -synuclein inherited in families afflicted with a clinical picture of parkinsonism and Lewy Body Dementia supplements recent structures of the wild type structure, and shows how a single residue change can result in a greatly changed structure that may underlie the inherited form of the disease.

biochemistry↗

bioRxiv2019

Neonatal brain-directed gene therapy rescues a mouse model of neurodegenerative CLN6 Batten disease

The neuronal ceroid lipofuscinoses (NCLs), more commonly referred to as Batten disease, are a group of inherited lysosomal storage disorders that present with neurodegeneration, loss of vision and premature death. There are at least 13 genetically distinct forms of NCL. Enzyme replacement therapies and preclinical studies on gene supplementation have shown promising results for NCLs caused by lysosomal enzyme deficiencies. The development of gene therapies targeting the brain for NCLs caused by defects in transmembrane proteins has been more challenging and only limited therapeutic effects in animal models have been achieved so far. Here, we describe the development of an adeno-associated virus (AAV)-mediated gene therapy to treat the neurodegeneration in a mouse model of CLN6 disease, a form of NCL with a deficiency in the membrane-bound protein CLN6. We show that neonatal bilateral intracerebroventricular injections with AAV9 carrying CLN6 increase lifespan by more than 90%, maintain motor skills and motor coordination and reduce neuropathological hallmarks of Cln6-deficient mice up to 23 months post vector administration. These data demonstrate that brain-directed gene therapy is a valid strategy to treat the neurodegeneration of CLN6 disease and may be applied to other forms of NCL caused by transmembrane protein deficiencies in the future.

neuroscience↗

bioRxiv2017

Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets

Control of metabolism by compartmentation is a widespread feature of higher cells. Recent studies have focused on dynamic intracellular bodies such as stress granules, P-bodies, nucleoli, and metabolic puncta. These bodies appear as separate phases, some containing reversible, amyloid-like fibrils formed by interactions of low-complexity protein domains. Here we report five atomic structures of segments of low-complexity domains from granule-forming proteins, one determined to 1.1 [A] resolution by micro-electron diffraction. Four of these interacting protein segments show common characteristics, all in contrast to pathogenic amyloid: kinked peptide backbones, small surface areas of interaction, and predominate attractions between aromatic side-chains. By computationally threading the human proteome on three of our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such reversible interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, keratins, and cornified envelope proteins, consistent with the capacity of cells to form a wide variety of dynamic intracellular bodies.\n\nOne Sentence SummaryAtomic structures show transient membraneless organelles of cells formed by a new type of protein interaction akin to pathogenic amyloid fibrils.

biochemistry↗