Jiang H, Cole PA. N-Terminal Protein Labeling with N-Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein-Protein Interactions Using Labeled Protein. Curr Protoc 2021;1(1):e14.Abstract
Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay.
Patnaik D, Pao P-C, Zhao W-N, Silva CM, Hylton NK, Chindavong PS, Pan L, Tsai L-H, Haggarty SJ. Exifone Is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration. ACS Chem Neurosci 2021;12(2):271-284.Abstract
Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.
Saundh SL, Patnaik D, Gagné S, Bishop JA, Lipsit S, Amat S, Pujari N, Nambisan AK, Bigsby R, Murphy M, Tsai L-H, Haggarty SJ, Leung AK-W. Identification and Mechanistic Characterization of a Peptide Inhibitor of Glycogen Synthase Kinase (GSK3β) Derived from the Disrupted in Schizophrenia 1 (DISC1) Protein. ACS Chem Neurosci 2020;11(24):4128-4138.Abstract
Glycogen synthase kinase 3-beta (GSK3β) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3β have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3β activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3β in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3β: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3β binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3β.
Lyons SM, Kharel P, Akiyama Y, Ojha S, Dave D, Tsvetkov V, Merrick W, Ivanov P, Anderson P. eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function. Nucleic Acids Res 2020;48(11):6223-6233.Abstract
As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.
Mineo M, Lyons SM, Zdioruk M, von Spreckelsen N, Ferrer-Luna R, Ito H, Alayo QA, Kharel P, Giantini Larsen A, Fan WY, Auduong S, Grauwet K, Passaro C, Khalsa JK, Shah K, Reardon DA, Ligon KL, Beroukhim R, Nakashima H, Ivanov P, Anderson PJ, Lawler SE, Chiocca AE. Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus. Mol Cell 2020;78(6):1207-1223.e8.Abstract
Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.
Lowey B, Whiteley AT, Keszei AFA, Morehouse BR, Mathews IT, Antine SP, Cabrera VJ, Kashin D, Niemann P, Jain M, Schwede F, Mekalanos JJ, Shao S, Lee ASY, Kranzusch PJ. CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection. Cell 2020;Abstract
cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.
Patsoukis N, Duke-Cohan JS, Chaudhri A, Aksoylar H-I, Wang Q, Council A, Berg A, Freeman GJ, Boussiotis VA. Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation. Commun Biol 2020;3(1):128.Abstract
Programmed cell death-1 (PD-1) inhibits T cell responses. This function relies on interaction with SHP-2. PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM) at Y223 and one immunoreceptor tyrosine-based switch motif (ITSM) at Y248. Only ITSM-Y248 is indispensable for PD-1-mediated inhibitory function but how SHP-2 enzymatic activation is mechanistically regulated by one PD-1 phosphotyrosine remains a puzzle. We found that after PD-1 phosphorylation, SHP-2 can bridge phosphorylated ITSM-Y248 residues on two PD-1 molecules via its amino terminal (N)-SH2 and carboxyterminal (C)-SH2 domains forming a PD-1: PD-1 dimer in live cells. The biophysical ability of SHP-2 to interact with two ITSM-pY248 residues was documented by isothermal titration calorimetry. SHP-2 interaction with two ITSM-pY248 phosphopeptides induced robust enzymatic activation. Our results unravel a mechanism of PD-1: SHP-2 interaction that depends only on ITSM-Y248 and explain how a single docking site within the PD-1 cytoplasmic tail can activate SHP-2 and PD-1-mediated inhibitory function.
Gorgulla C, Boeszoermenyi A, Wang Z-F, Fischer PD, Coote P, Padmanabha Das KM, Malets YS, Radchenko DS, Moroz YS, Scott DA, Fackeldey K, Hoffmann M, Iavniuk I, Wagner G, Arthanari H. An open-source drug discovery platform enables ultra-large virtual screens. Nature 2020;Abstract
On average, an approved drug today costs $2-3 billion and takes over ten years to develop. In part, this is due to expensive and time-consuming wet-lab experiments, poor initial hit compounds, and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening (SBVS) has the potential to mitigate these problems. With SBVS, the quality of the hits improves with the number of compounds screened. However, despite the fact that large compound databases exist, the ability to carry out large-scale SBVSs on computer clusters in an accessible, efficient, and flexible manner has remained elusive. Here we designed VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large ligand libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we have prepared the largest and freely available ready-to-dock ligand library available, with over 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened over 1 billion compounds and discovered a small molecule inhibitor (iKeap1) that engages KEAP1 with nanomolar affinity (K = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. We also identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify binders with high affinity for target proteins.
Patnaik D, Pao P-C, Zhao W-N, Silva CM, Hylton NK, Chindavong PS, Pan L, Tsai L-H, Haggarty SJ. Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration. bioRxiv 2020;27(2):2020.03.02.973636.Abstract
Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative disease. Preventing this loss of genomic integrity that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent, class I histone deacetylase HDAC1 has been identified as a critical protein for protecting neurons from deleterious effects mainly caused by double-strand DNA breaks in Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS or FTD will benefit from the identification of small molecules capable of selectively increasing the deacetylase activity of HDAC1 over other structurally similar class I HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive decline associated with AD and Parkinson’s disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1 and provides protection against genotoxic stress. We show that exifone acts as a mixed, non-essential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Selectivity profiling and estimation of kinetic parameters using biolayer interferometry suggest HDAC1 is a preferential target compared to other class I HDACs and CDK5. Treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in a decrease of histone acetylation, consistent with an intracellular mechanism of deacetylase activation. Moreover, using tauopathy patient-derived iPSC neuronal models subject to oxidative stress through mitochondrial inhibition exifone treatment was neuroprotective. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.
Bernard-Gauthier V, Mossine AV, Knight A, Patnaik D, Zhao W-N, Cheng C, Krishnan HS, Xuan LL, Chindavong PS, Reis SA, Chen JM, Shao X, Stauff J, Arteaga J, Sherman P, Salem N, Bonsall D, Amaral B, Varlow C, Wells L, Martarello L, Patel S, Liang SH, Kurumbail RG, Haggarty SJ, Scott PJH, Vasdev N. Structural Basis for Achieving GSK-3β Inhibition with High Potency, Selectivity, and Brain Exposure for Positron Emission Tomography Imaging and Drug Discovery. J Med Chem 2019;62(21):9600-9617.Abstract
Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3β-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2 and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.
Rovere M, Powers AE, Jiang H, Pitino JC, Fonseca-Ornelas L, Patel DS, Achille A, Langen R, Varkey J, Bartels T. E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity. J Biol Chem 2019;294(25):9799-9812.Abstract
Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region ( E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA-responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other "synucleinopathies." In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle-like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.
Watanabe A, McCarthy KR, Kuraoka M, Schmidt AG, Adachi Y, Onodera T, Tonouchi K, Caradonna TM, Bajic G, Song S, McGee CE, Sempowski GD, Feng F, Urick P, Kepler TB, Takahashi Y, Harrison SC, Kelsoe G. Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism. Cell 2019;177(5):1124-1135.e16.Abstract
Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.
Rogers JM, Waters CT, Seegar TCM, Jarrett SM, Hallworth AN, Blacklow SC, Bulyk ML. Bispecific Forkhead Transcription Factor FoxN3 Recognizes Two Distinct Motifs with Different DNA Shapes. Mol Cell 2019;74(2):245-253.e6.Abstract
Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition.
Pitts J, Hsia C-Y, Lian W, Wang J, Pfeil M-P, Kwiatkowski N, Li Z, Jang J, Gray NS, Yang PL. Identification of small molecule inhibitors targeting the Zika virus envelope protein. Antiviral Res 2019;164:147-153.Abstract
The recent emergence of Zika virus, a mosquito-borne flavivirus, in the Americas has shed light on the severe neurological diseases associated with infection, notably congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Despite the recent focus on Zika virus, there are currently no approved vaccines or antiviral therapies available to treat or prevent infection. In this study we established a competitive amplified luminescent proximity homogeneous assay (ALPHAscreen) to identify small molecule inhibitors targeting the envelope protein of Zika virus (Zika E). We utilized this assay to screen two libraries of nearly 27,000 compounds and identified seven novel inhibitors of Zika E. Characterization of these primary screening leads demonstrated that inhibition of Zika virus occurs at non-cytotoxic concentrations for all seven lead compounds. In addition, we found that all seven lead compounds have potent activity against the closely related dengue virus 2 but not vesicular stomatitis virus, an unrelated enveloped virus. Biochemical experiments indicate that these compounds act by preventing E-mediated membrane fusion. This work highlights a new method for the discovery and optimization of direct-acting antivirals targeting the E protein of Zika and other flaviviruses.
Silva CM, Ferguson FM, Cai Q, Donovan KA, Nandi G, Patnaik D, Zhang T, Huang H-T, Lucente DE, Dickerson BC, Mitchison TJ, Fischer ES, Gray NS, Haggarty SJ. Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. Elife 2019;8Abstract
Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01-175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4, to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01-175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated -knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.
Li P-C, Jang J, Hsia C-Y, Groomes PV, Lian W, de Wispelaere M, Pitts JD, Wang J, Kwiatkowski N, Gray NS, Yang PL. Small Molecules Targeting the Flavivirus E Protein with Broad-Spectrum Activity and Antiviral Efficacy in Vivo. ACS Infect Dis 2019;5(3):460-472.Abstract
Vaccines and antivirals to combat dengue, Zika, and other flavivirus pathogens present a major, unmet medical need. Vaccine development has been severely challenged by the antigenic diversity of these viruses and the propensity of non-neutralizing, cross-reactive antibodies to facilitate cellular infection and increase disease severity. As an alternative, direct-acting antivirals targeting the flavivirus envelope protein, E, have the potential to act via an analogous mode of action without the risk of antibody-dependent enhancement of infection and disease. We previously discovered that structurally diverse small molecule inhibitors of the dengue virus E protein exhibit varying levels of antiviral activity against other flaviviruses in cell culture. Here, we demonstrate that the broad-spectrum activity of several cyanohydrazones against dengue, Zika, and Japanese encephalitis viruses is due to specific inhibition of E-mediated membrane fusion during viral entry and provide proof of concept for pharmacological inhibition of E as an antiviral strategy in vivo.
Wu AR, Semlow DR, Kamimae-Lanning AN, Kochenova OV, Chistol G, Hodskinson MR, Amunugama R, Sparks JL, Wang M, Deng L, Mimoso CA, Low E, Patel KJ, Walter JC. TRAIP is a master regulator of DNA interstrand crosslink repair. Nature 2019;567(7747):267-272.Abstract
Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.
Zheng S, Abreu N, Levitz J, Kruse AC. Structural basis for KCTD-mediated rapid desensitization of GABA signalling. Nature 2019;567(7746):127-131.Abstract
The GABA (γ-aminobutyric acid type B) receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors, including G-protein-coupled inwardly rectifying potassium channels (GIRKs). GABA-receptor signalling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization. However, the mechanistic basis for KCTD modulation of GABA signalling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, and functional and biochemical experiments, we reveal the molecular details of KCTD binding to both GABA receptors and G-protein βγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor carboxy-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gβγ in which the G-protein subunits also interact directly with one another. We further show that KCTD binding to Gβγ is highly cooperative, defining a model in which KCTD proteins cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABA signalling by KCTD proteins.
Rovere M. Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes. Methods Mol Biol 2019;1948:123-143.Abstract
α-Synuclein's physiology and pathology have been linked by numerous reports to its ability to bind and remodel membranes, especially at synaptic terminals. It is therefore critical for researchers investigating the determinants of these interactions to rely on methods capable of providing an accurate and complete physicochemical snapshot of the binding events. Circular dichroism (CD) and isothermal titration calorimetry (ITC) are established techniques for the study of binding equilibria in biological systems and, especially when used in combination, allow a thorough characterization of the protein-lipid interplay.Here we provide general guidelines and describe some common pitfalls of these experiments. This protocol describes the preparation of small unilamellar vesicles (SUVs), mimicking the curved bilayers α-synuclein normally interacts with, the CD-monitored titration of α-synuclein with SUVs, the ITC (lipid-into-protein) experiment, and the subsequent data analysis using an n independent binding site model.
Wang L, Fu T-M, Zhou Y, Xia S, Greka A, Wu H. Structures and gating mechanism of human TRPM2. Science 2018;362(6421)Abstract
Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain responsible for binding adenosine diphosphate (ADP)-ribose (ADPR), and both ADPR and calcium (Ca) are required for TRPM2 activation. Here we report cryo-electron microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM homology region (MHR1/2/3) in the apo state but undergoes conformational changes upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the intersubunit interaction. The binding of Ca further engages transmembrane helices and the conserved TRP helix to cause conformational changes at the MHR arm and the lower gating pore to potentiate channel opening. These findings explain the molecular mechanism of concerted TRPM2 gating by ADPR and Ca and provide insights into the gating mechanism of other TRP channels.