Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)₂ that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)₂ associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5′-to-5′ arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3′-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.     

Solution structure of a DNA quadruplex containing ALS and FTD related GGGGCC repeat stabilized by 8-bromodeoxyguanosine substitution

A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K⁺ ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G₄C₂)₃G₄] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G₄C₂)₃GG^BrGG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.     

Loop lengths of G-quadruplex structures affect the G-quadruplex DNA binding selectivity of the RGG motif in Ewing's sarcoma

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro. Here, we show that the RGG of EWS binds preferentially to G-quadruplexes with longer loops, which is not related to the topology of the G-quadruplex structure. Moreover, the G-quadruplex DNA binding of the RGG in EWS depends on the phosphate backbone of the loops in the G-quadruplex DNA. We also investigated the G-quadruplex DNA binding activity of the N- and C-terminally truncated RGG to assess the role of the regions in the RGG in G-quadruplex DNA binding. Our findings indicate that the RGG and the other arginine-rich motif of residues 617-656 of the RGG in EWS are important for the specific binding to G-quadruplex DNA. These findings will contribute to the development of molecules that selectively target different G-quadruplex DNA.     

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat‐associated non‐AUG (RAN) translation of repeat‐containing C9orf72 RNA results in the production of neurotoxic dipeptide‐repeat proteins (DPRs). Here, we developed a high‐throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP‐elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA‐catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient‐derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.     

Development of a specific live-cell assay for native autophagic flux

Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high-throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.     

A new cationic porphyrin derivative (TMPipEOPP) with large side arm substituents: a highly selective G-quadruplex optical probe

The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA) sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4), interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1-piperidinyl)ethoxy]phenyl} porphyrin (TMPipEOPP), with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.     

Protection of DNA ends by telomeric 3' G-tail sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.     

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3′-end tetrad of a DNA G-quadruplex

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) – a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3′-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5′-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.     

Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation

There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes with specificity. Such molecules would be useful to test hypotheses for telomere length regulation, and may have therapeutic potential. The versatility and modular nature of the zinc finger motif makes it an ideal candidate for engineering G-quadruplex-binding proteins. Phage display technology has previously been widely used to screen libraries of zinc fingers for binding to novel duplex DNA sequences. In this study, a three-finger library has been screened for clones that bind to an oligonucleotide containing the human telomeric repeat sequence folded in the G-quadruplex conformation. The selected clones show a strong amino acid consensus, suggesting analogous modes of binding. Binding was found to be both sequence dependent and structure specific. This is the first example of an engineered protein that binds to G-quadruplex DNA, and represents a new type of binding interaction for a zinc finger protein.     

Selective recognition of a DNA G-quadruplex by an engineered antibody

Particular guanine rich nucleic acid sequences can fold into stable secondary structures called G-quadruplexes. These structures have been identified in various regions of the genome that include the telomeres, gene promoters and UTR regions, raising the possibility that they may be associated with biological function(s). Computational analysis has predicted that intramolecular G-quadruplex forming sequences are prevalent in the human genome, thus raising the desire to differentially recognize genomic G-quadruplexes. We have employed antibody phage display and competitive selection techniques to generate a single-chain antibody that shows >1000-fold discrimination between G-quadruplex and duplex DNA, and furthermore >100-fold discrimination between two related intramolecular parallel DNA G-quadruplexes. The amino acid sequence composition at the antigen binding site shows conservation within the light and heavy chains of the selected scFvs, suggesting sequence requirements for G-quadruplex recognition. Circular dichroism (CD) spectroscopic data showed that the scFv binds to the prefolded G-quadruplex and does not induce G-quadruplex structure formation. This study demonstrates the strongest discrimination that we are aware of between two intramolecular genomic G-quadruplexes.     

Dysregulated molecular pathways in amyotrophic lateral sclerosis–frontotemporal dementia spectrum disorder

Frontotemporal dementia (FTD), the second most common form of dementia in people under 65 years of age, is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD overlaps extensively with the motor neuron disease amyotrophic lateral sclerosis (ALS), especially at the genetic level. Both FTD and ALS can be caused by many mutations in the same set of genes; the most prevalent of these mutations is a GGGGCC repeat expansion in the first intron of C9ORF72. As shown by recent intensive studies, some key cellular pathways are dysregulated in the ALS‐FTD spectrum disorder, including autophagy, nucleocytoplasmic transport, DNA damage repair, pre‐mRNA splicing, stress granule dynamics, and others. These exciting advances reveal the complexity of the pathogenic mechanisms of FTD and ALS and suggest promising molecular targets for future therapeutic interventions in these devastating disorders.