G‐Quadruplexes act as sequence‐dependent protein chaperones
2020; Springer Nature; Volume: 21; Issue: 10 Linguagem: Inglês
10.15252/embr.201949735
ISSN1469-3178
AutoresAdam Begeman, Ahyun Son, Theodore J. Litberg, Tadeusz Wroblewski, Thane Gehring, Veronica Huizar Cabral, Jennifer N. Bourne, Zhenyu Xuan, Scott Horowitz,
Tópico(s)DNA Repair Mechanisms
ResumoArticle18 September 2020Open Access Source DataTransparent process G-Quadruplexes act as sequence-dependent protein chaperones Adam Begeman Adam Begeman orcid.org/0000-0001-5458-8610 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Ahyun Son Ahyun Son orcid.org/0000-0002-7979-0409 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Theodore J Litberg Theodore J Litberg orcid.org/0000-0001-5353-8451 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Tadeusz H Wroblewski Tadeusz H Wroblewski Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Thane Gehring Thane Gehring Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Veronica Huizar Cabral Veronica Huizar Cabral Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Jennifer Bourne Jennifer Bourne Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA Search for more papers by this author Zhenyu Xuan Zhenyu Xuan Department of Biological Sciences, Center for Systems Biology, University of Texas at Dallas, Richardson, TX, USA Search for more papers by this author Scott Horowitz Corresponding Author Scott Horowitz [email protected] orcid.org/0000-0002-1148-0105 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Adam Begeman Adam Begeman orcid.org/0000-0001-5458-8610 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Ahyun Son Ahyun Son orcid.org/0000-0002-7979-0409 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Theodore J Litberg Theodore J Litberg orcid.org/0000-0001-5353-8451 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Tadeusz H Wroblewski Tadeusz H Wroblewski Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Thane Gehring Thane Gehring Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Veronica Huizar Cabral Veronica Huizar Cabral Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Jennifer Bourne Jennifer Bourne Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA Search for more papers by this author Zhenyu Xuan Zhenyu Xuan Department of Biological Sciences, Center for Systems Biology, University of Texas at Dallas, Richardson, TX, USA Search for more papers by this author Scott Horowitz Corresponding Author Scott Horowitz [email protected] orcid.org/0000-0002-1148-0105 Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA Search for more papers by this author Author Information Adam Begeman1,‡, Ahyun Son1,‡, Theodore J Litberg1, Tadeusz H Wroblewski1, Thane Gehring1, Veronica Huizar Cabral1, Jennifer Bourne2, Zhenyu Xuan3 and Scott Horowitz *,1 1Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA 2Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA 3Department of Biological Sciences, Center for Systems Biology, University of Texas at Dallas, Richardson, TX, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 303 871 4326; E-mail: [email protected] EMBO Reports (2020)21:e49735https://doi.org/10.15252/embr.201949735 See also: J Aarum et al (October 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Maintaining proteome health is important for cell survival. Nucleic acids possess the ability to prevent protein aggregation more efficiently than traditional chaperone proteins. In this study, we explore the sequence specificity of the chaperone activity of nucleic acids. Evaluating over 500 nucleic acid sequences’ effects on protein aggregation, we show that the holdase chaperone effect of nucleic acids is sequence-dependent. G-Quadruplexes prevent protein aggregation via quadruplex:protein oligomerization. They also increase the folded protein level of a biosensor in E. coli. These observations contextualize recent reports of quadruplexes playing important roles in aggregation-related diseases, such as fragile X and amyotrophic lateral sclerosis (ALS), and provide evidence that nucleic acids have the ability to modulate the folding environment of E. coli. Synopsis Examining nucleic acids for the sequence dependence of their chaperone activity finds quadruplexes have potent roles in protein oligomerization and aggregation prevention. Chaperone nucleic acid activity is sequence dependent. Quadruplexes modulate protein oligomerization. Chaperone nucleic acid activity can occur in E. coli. Introduction Chaperones are a diverse group of proteins and other molecules that regulate proteostasis (Hartl et al, 2011) in the cell by preventing protein aggregation (holdases) and helping protein folding (foldases). Recently, molecules other than traditional protein chaperones have been shown to play important roles in these processes (Gray et al, 2014; Ray, 2017). We recently showed that nucleic acids can possess potent holdase activity, with the best sequences having higher holdase activity than any previously characterized chaperone (Docter et al, 2016). Nucleic acids can also collaborate with Hsp70 to help protein folding, acting similarly to small heat shock proteins (Jakob et al, 1993, 1999; Haslbeck & Vierling, 2015; Docter et al, 2016). Nucleic acids can also bring misfolded proteins to stress granules (Bounedjah et al, 2014) and are a primary component of the nucleolus, which was recently shown to store misfolded proteins under stress conditions (Frottin et al, 2019). However, the structural characteristics, sequence dependence, and mechanistic understanding of how nucleic acids act as chaperones remain unclear. A critical question in understanding the holdase activity of nucleic acids is whether this activity is sequence specific? Previously, we showed that polyA, polyT, polyG, and polyC prevented aggregation with varying kinetics, suggesting that sequence specificity is possible (Docter et al, 2016). Here, we tested sequence specificity by examining over 500 nucleic acids of varying sequence for holdase activity. The holdase activity is found to be highly sequence specific, with quadruplexes showing the greatest activity. Several quadruplexes displayed generality, with potent holdase activity for a variety of different proteins. This activity was also verified to occur in Escherichia coli. Further examination of these quadruplex sequences demonstrated that the holdase activity largely arises through quadruplex:protein oligomerization. These results help explain several recent reports of quadruplex sequences playing important roles in oligomerization, aggregation, and phase separation in biology and pathology, and that these are common properties of quadruplex interactions with partially unfolded or disordered proteins. They also represent a strong demonstration of the ability of nucleic acids in modulating protein folding in E. coli. Results Sequence specificity of holdase activity To determine the sequence specificity of the holdase activity of nucleic acids, we measured light scattering and turbidity via absorbance in a thermal aggregation assay (Fig 1A) for 312 nucleic acid sequences (Fig 1B). These nucleic acids were nearly all 20 bases in length, single-stranded DNA (ssDNA) sequences of random composition. Bulk DNA was used as a positive control (Docter et al, 2016). Plotting the percent aggregation for each sequence demonstrates that the holdase activity of the ssDNA is sequence-dependent (Fig 1B). Sequences nearly spanned the complete range in activity, from barely affecting aggregation, to nearly preventing all protein aggregation for over an hour. Figure 1. Testing Sequence Dependence of Chaperone Nucleic Acid Activity A. Representative example citrate synthase protein aggregation assay. Turbidity and light scattering were measured in a multimode plate reader at 360 nm for 1.5 h of incubation at 50°C. Blue lines represent triplicate citrate synthase alone, red and orange lines represent triplicate citrate synthase incubated with a single ssDNA sequence, green is buffer alone. B. Screen of ssDNA sequences for holdase chaperone activity. Each bar represents a different 20-nt sequence, sorted by activity. Aggregation % was measured as the normalized average of triplicate (technical replicate) citrate synthase turbidity measurements after 1.5 h of incubation at 50°C (representative example shown in Fig 1A). Lower aggregation indicates greater holdase function. The initial screen used random, non-redundant sequences (top), which led to a follow-up enriched screen (bottom). Error bars are mean ± SE. C. HOMER Logo of motif found by analyzing screen (statistics using a binomial distribution with the default setting by HOMER to calculate P-value of motif enrichment (Benner et al, 2017): P < 1.0 × 10−13, FDR < 0.001, % of Targets: 53.85, % of Background: 7.69). Source data are available online for this figure. Source Data fore Figure 1 [embr201949735-sup-0004-SDataFig1.zip] Download figure Download PowerPoint With this high level of sequence dependence, we next performed bioinformatics to determine if any sequence motifs encoded holdase activity. We first found that the holdase activity is positively correlated with the guanine content in the sequences (ρ = 0.24, P-value = 1.5 × 10−5). Comparing the top third in holdase activity to the bottom third, only one motif was found to be significantly enriched in sequences with higher holdase activity (53.85% vs. 7.69%, FDR = 0.001; Fig 1C). This motif contains five consecutive guanines followed by any base and then thymine. A similar guanine-rich (G-rich) motif (consensus pattern: BGGSTGAT) was also found by a regression-based method (R2 = 0.61, P-value = 1 × 10−5). This analysis suggests that the most potent holdase activity was encoded by a polyG motif. To verify this polyG motif, we tested another 192 sequences for holdase activity that had high guanine content. These sequences include 96 sequences with a 55% bias toward guanine bases, 40 sequences with a 75% bias toward guanine bases, and 56 having different positional variations of the aforementioned polyG motif (Fig 1C). Comparing the original random sequences to these G-rich sequences, the average aggregation was substantially reduced in the enriched guanine set, from 64.8% to 32.0%. Within the enriched guanine set, however, there was still a great deal of variation, with the data spanning aggregation from 72% to 4%. This wide variability suggests that the motif requires more than just the high guanine content. Within the subset of sequences with a 55% bias toward guanine, a significant polyG motif was again identified by comparing sequences having different holdase activity. Within the subset of 75% guanine-containing sequences, no statistically significant differences were found, as most sequences contained at least one polyG motif. We also tested holdase activity for 56 sequences having different positional variations of the aforementioned polyG motif, which did not find positional dependency within the sequence for the holdase activity. In the best sequences from this enriched assay, the nucleic acid completely prevented protein aggregation for the entire hour and a half experiment. These results confirmed that the holdase activity is associated with a polyG motif. We further characterized the holdase activity of the polyG-containing sequences using chemical denaturation aggregation assays in which the protein starts in a denatured state. Light scattering experiments confirmed the holdase activity in at least nine different polyG-containing sequences (Figs 2A and EV1, and Table 1). These data suggest that the polyG-containing sequences preferentially bind to a misfolded or partially denatured form of the protein rather than the native state. Choosing three of these sequences for further investigation (sequences 359, 536, and 576, Table 1), we performed concentration dependent assays and found that all three displayed strong concentration dependence in their activity (Fig 2B and C), unlike a control sequence with no polyG motif (sequence 42, Table 1), which did not display strong activity in our initial screen. Figure 2. Concentration dependence of chaperone nucleic acid activity A. Citrate Synthase aggregation from chemically induced denaturation via right angle light scattering at 360 nm. Sequences 359, 536, and 576 all displayed holdase activity and contain a polyG motif. Sequence 42 was used as a negative control, as it performed poorly as a holdase chaperone and did not contain a polyG motif. B. Percent aggregation in thermal denaturation assay with varying concentrations of select quadruplex-forming sequences (Sequences 359, 536, and 576) and negative control sequence (Seq42). Concentrations are ssDNA strand to protein ratios of: 0.5:1, 1:1, 2:1, 4:1, and 8:1. Error bars are mean ± SE of technical triplicates. C. Citrate Synthase aggregation from chemically induced denaturation via right angle light scattering at 360 nm as a function of concentration of Sequence 359. Source data are available online for this figure. Source Data fore Figure 2 [embr201949735-sup-0005-SDataFig2.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Representative examples of chemical aggregation tests of multiple quadruplex-containing sequences with citrate synthaseRepresentative examples of right angle light scattering of chemically denatured citrate synthase with quadruplex-containing sequences measured via fluorimeter. DNA strand to protein ratio is 2:1. Download figure Download PowerPoint Table 1. Select DNA sequences used in this study Name Sequence Aggregation Assays in Figures Quadruplex? (G4 Hunter Score or reference) Seq359 GGG GGG GTA ACG GGC TGG TT Figs 2 and 4 (SeqAa), EV1, EV4 Yes (1.95) Seq536 GAG GGG GGC TGC CGT TCA CA Figs 2 and 4 (SeqFa), EV1 Yes (1.00) Seq576 TGT CGG GCG GGG AGG GGG GG Figs 2 and 4 (SeqJa), Fig 4, EV1 Yes (2.60) Seq42 AAC GAA AGA ACA TAA TCT CG Figs 2, EV1 No (< 0.1) Seq398 GGG GGG CGG TGC GGT AGC GA Fig 4 (SeqBa) Yes (1.60) Seq567 GGG GGG GCG GCG GGG GGG AG Fig 4 (SeqCa) Yes (2.95) Seq361 CGG ATG GGG TGG GTG CTG GA Fig 4 (SeqDa) Yes (1.60) Seq573 GGC GGG CGG TGG GGG GTG CG Fig 4 (SeqEa), EV1 Yes (2.00) Seq563 TAG GTG GGA GGT GCG GGA GG Fig 4 (SeqGa) Yes (1.50) Seq357 ATG AGT TGG TGC GTG GGG GA Fig 4 (SeqHa) Yes (1.35) Seq345 GGG GTT GGT GGG GGG GTA TA Fig 4 (SeqIa) Yes (2.40) Seq582 GGG GGG GAC GGT GGC GAG GG Fig 4 (SeqKa) Yes (2.20) Seq592 GGG GGG GGG GCC GGG GGG GT Fig 4 (SeqLa), EV1 Yes (3.20) Seq579 GGA GGG GGG GGG GTG AGG GG Fig 4 (SeqMa) Yes (3.05) Seq353 CGG CCG GGC GGG GTC CGG TT Fig 4 (SeqNa) Yes (1.15) Seq364 GGG CTT TGC ATT TCT ATG GT Fig 4 (SeqOa) No (0.55) Seq63 GTG GGA TGT CAG ACG TGG AC Fig 4 (SeqPa) No (0.7) Seq60 CAT CCG AGG TTT ACT CCC CC Fig 4 (SeqQa) No (−1.05) Seq185 AAA CGT GCA GTG CAA CAT AA Fig 4 (SeqRa) No (< 0.1) Seq190 GTA CTT TTG GCA TCC TCA CA Fig 4 (SeqSa) No (−0.15) Seq259 AGT CTT GTT GTG ACT CAA CT Fig 4 (SeqTa) No (< 0.1) Seq305 GAT GAT GTC CGT AGC TTG CC Fig 4 (SeqUa) No (−0.15) Seq347 AAT GGG ATG CCA TTT GCT GG Fig 4 (SeqVa) No (0.5) Seq209 GAT ATA GCT GGA GTA CAA CC Fig 4 (SeqWa) No (< 0.1) Seq205 CCA CGA CTG CAG AGG TAT GT Fig 4 (SeqXa) No (< 0.1) Seq340 GGT AGT TCG GTT GGT GGG GA Fig EV1 Yes (1.40) Seq589 GCG GGG GGA GGG AGG AGG GG Fig EV1 Yes (2.65) Seq580 CGG GGG TGG AGG GGG GGG AG Fig EV1 Yes (2.80) Seq583 CGG GAA GGG GGG GCG GAG GG Fig EV1 Yes (2.40) Basic Anti-Parallel GGG GTT TTG GGG Fig 3 Yes ref: (Haider et al, 2002) Thrombin Binding Aptamer (TBA) GGT TGG TGT GGT TGG Fig 3 Yes ref: (Macaya et al, 1993) Core Human Telomer Quadruplex AGG TTA GGG TTA GGG TTA GGG Fig 3 Yes ref: (Renčiuk et al, 2009) Wild Type c-MYC TGA GGG TGG GGA GGG TGG GGA AGG Fig 3 Yes ref: (Simonsson et al, 1998; Phan et al, 2004; Ambrus et al, 2005) MYC22 TGA GGG TGG GGA GGG TGG GGA A Fig 3 Yes ref: (Phan et al, 2004; Ambrus et al, 2005) MYC12 TGG GGA GGG TTT TTA GGG TGG GGA Fig 3 Yes ref: (Phan et al, 2004) PARP1 Promoter TGG GGG CCG AGG CGG GGC TTG GG Fig 3 Yes ref: (Sengar et al, 2019) LTR-III GGG AGG CGT GGC CTG GGC GGG ACT GGG G Fig 3 Yes ref: (Butovskaya et al, 2018) For the full list of sequences used, see the supplemental data file. a Lettering from left to right in figure. G-Quadruplexes as potent holdases PolyG is well known to form quadruplexes when provided with appropriate counter ions. Composed of polyG bases forming pi-stacked tetrads, guanine quadruplexes are a class of structured nucleic acids that have been of increasing interest due to their regulatory role in replication, transcription, and translation (Paeschke et al, 2005; Rhodes & Lipps, 2015). Quadruplexes have also recently been implicated in several protein aggregation genetic disorders, such as fragile X syndrome and ALS (DeJesus-Hernandez et al, 2011; Zhang et al, 2014; Stefanovic et al, 2015; Vasilyev et al, 2015; Afroz et al, 2017; Balendra & Isaacs, 2018). To test if the sequences containing polyG that had potent holdase activity were forming quadruplexes, we performed circular dichroism (CD) spectroscopy experiments on sequences 359, 536, and 576 to determine their secondary structure. The CD spectra showed distinct peaks at 260 and 210 nm, with a trough at 245 nm, indicative of parallel quadruplex formation (Fig 3A; Paramasivan et al, 2007). This pattern was not observed in sequence 42, which had no polyG motif, and was therefore not expected to form a quadruplex. This supposition was further supported by examining the emission spectra of N-methylmesoporphyrin IX (NMM), a well-characterized parallel quadruplex-binding fluorophore (Sabharwal et al, 2014; Manna & Srivatsan, 2018). Due to the sensitivity limitations of CD, we examined the fluorescence spectra of NMM in the presence of 1 μM DNA, which was the same concentration used in the holdase assays. The NMM spectra indicate that all three sequences form parallel quadruplexes at the concentration used in aggregation assays, unlike the ssDNA control (Fig 3B). Figure 3. Characterization of quadruplex content and holdase activity A. Structural characterization of holdase nucleic acids using circular dichroism in sodium phosphate buffer. Peaks are observed at 260 nm and 210 nm, as well as a trough at 245 nm, indicating the presence of parallel G-quadruplexes. Thermal stability of quadruplexes shown in Fig EV2 in potassium phosphate buffer. B. NMM fluorescence measured at 610 nm. Error bars are mean ± SE of technical triplicates. C. Comparing holdase activity of different quadruplex-containing sequences of known topology (Sundquist & Klug, 1989; Macaya et al, 1993; Simonsson et al, 1998; Haider et al, 2002; Phan et al, 2004; Ambrus et al, 2005; Renčiuk et al, 2009; Butovskaya et al, 2018; Sengar et al, 2019). Error bars are mean ± SE of technical triplicate. Source data are available online for this figure. Source Data fore Figure 3 [embr201949735-sup-0006-SDataFig3.xlsx] Download figure Download PowerPoint Circular dichroism melting experiments indicate that these quadruplex structures are stable. At 45°C, > 90% of the quadruplex structures remained for sequences 536 and 576, and 70% for sequence 359 (Fig EV2). Click here to expand this figure. Figure EV2. CD spectra of select 20mers (DNA (A) and (B), and RNA (C) and (D)) in 10 mM pH 7.5 potassium phosphateThe use of potassium phosphate here was to better mimic the initial aggregation screen in which potassium was used in the HEPES buffer. A, C. The thermal stability of quadruplex-containing sequences as measured by CD spectroscopy where each line represents a wavelength scan at the indicated temperature. B, D. The secondary structure of the same quadruplex-containing sequences at 25°C prior to annealing or after annealing at 25°C. Download figure Download PowerPoint We also checked whether RNA versions of these sequences would also form quadruplexes. For 536 and 576, the conformation was similar for both DNA and RNA, with all spectra indicating parallel quadruplex formation. However, for sequence 359, the type and stability of quadruplex conformation depended on the type of nucleic acid present. The DNA version at 45°C had spectra showing a mixture of parallel and anti-parallel quadruplex. The RNA version appeared to be more stable, with 80% of the original quadruplex structure remaining at 45°C. Furthermore, the spectra indicate that only the parallel quadruplex is formed in the case of sequence 359 RNA. The change in structure for 359 from a mixed topology to parallel merited separate testing of its chaperone activity. Heat denaturation assays demonstrate that the RNA version also had potent chaperone activity, preventing aggregation even more than its DNA counterpart (Fig EV3). Click here to expand this figure. Figure EV3. Comparison of holdase activity of RNA and DNA counterparts of Sequence 359 using citrate synthase heat denaturationData presented as means ± SE (n = 3) of technical triplicates. Source data are available online for this figure. Download figure Download PowerPoint These experiments confirmed that the holdase activity of these polyG-containing sequences is associated with quadruplex structure. Re-analyzing the heat denaturation aggregation assay data presented above, of the 160 sequences tested that had a polyG motif, 133 appeared in the top third of data, making up 79% of the sequences in that subset. 152 of the 160 polyG sequences also decreased aggregation by at least 50%. The higher level of activity from quadruplex DNA raised the question of whether any structured DNA could have a similar effect. In other words, could the activity arise from any DNA with greater structure than ssDNA? To test this possibility, we tested the holdase activity of 24 duplexed sequences to compare directly with their single-stranded counterparts. Overall, the differences were small, and in many cases statistically insignificant (Fig EV4). These experiments suggest that the holdase activity displayed here could be specific to quadruplex structures, and not to other structured DNAs. Click here to expand this figure. Figure EV4. Holdase activity of ssDNA toward citrate synthase compared to its duplexed counterpart as measured by heat denaturationData presented as mean ± SE (n = 3) of technical triplicate. Source data are available online for this figure. Download figure Download PowerPoint A related question was whether different forms of quadruplex structures have different intrinsic aggregation or chaperone tendencies. To test this question, we chose sequences that had previously determined quadruplex structures (Sundquist & Klug, 1989; Macaya et al, 1993; Simonsson et al, 1998; Haider et al, 2002; Phan et al, 2004; Ambrus et al, 2005; Renčiuk et al, 2009; Butovskaya et al, 2018; Sengar et al, 2019), and that were ∼20 bases in length and subjected them to the heat denaturation aggregation experiment. The different sequences displayed varying activities that appeared to correlate with structural properties (Fig 3C). Namely, anti-parallel quadruplexes appeared to have no chaperone activity or appeared to increase protein aggregation, and parallel quadruplexes had minor chaperone activity, but 3 + 1 mixed quadruplexes displayed greater chaperone activity. These results suggest that the type of quadruplex matters in its chaperone function, along with adjacent sequences, consistent with the observations of our best tested sequence, 359. Generality of holdase activity To determine the generality of this quadruplex holdase activity, we performed aggregation assays with three other proteins, luciferase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH). To check whether this holdase activity is quadruplex-specific with multiple proteins, we tested 14 sequences predicted to form quadruplexes by G4Hunter and 10 single-stranded sequences with each protein (Table 1). These proteins have varying structural properties, ranging in pI from 6.1 to 8.5, and size from 62.9 kD to 140 kD. With all four proteins, the quadruplexes severely decreased protein aggregation, demonstrating strong holdase activity. For luciferase, LDH, and MDH, most of the quadruplex sequences tested were able to completely prevent protein aggregation. However, the single-stranded sequences demonstrated little to no activity for all of these proteins (Fig 4). These data strongly suggest that the holdase activity displayed by quadruplex sequences is general, while also unique to quadruplex-forming sequences. Of note, LDH is the only of these proteins with previously characterized DNA-binding activity toward both duplex and ssDNA (Cattaneo et al, 1985; Grosse et al, 1986; Fang et al, 2000), but its aggregation was only significantly reduced by binding to quadruplex sequences (Fig 4). Of note, two sequences (O and P) that G4Hunter did not predict as having high quadruplex probability but had significant holdase activity, do have substantial guanine content and could potentially still form quadruplexes despite being listed as ssDNA here. Figure 4. Generality of G-quadruplex holdase activity using four different proteinsLuciferase (Luc), Citrate Synthase (CS), L-Malate Dehydrogenase (MDH), and L-Lactate Dehydrogenase (LDH) boxed in red are the 14 sequences with the propensity to form quadruplexes (as identified by G4Hunter), while the remaining 10 sequences are non-structured ssDNA (left). These data were also shown as a heat map (right). Error bars are mean ± SE of technical triplicates. Source data are available online for this figure. Source Data fore Figure 4 [embr201949735-sup-0007-SDataFig4.xlsx] Download figure Download PowerPoint Chaperone activity in Escherichia coli With our newfound quadruplex-containing sequences possessing powerful holdase activity in vitro, we sought to test whether they would have chaperone-like activity in a cellular system. For these experiments, we assayed the ability of these nucleic acids to improve the folding of fluorescent proteins in E. coli. GFP's fluorescence is dependent almost solely on its folding to its native state, which allows self-catalyzed chromophore formation, and continued maintenance of the native state causes continued fluorescence (Craggs, 2009). Furthermore, although many variants of GFP were later engineered to have fast maturation times and high stability, wildtype GFP (wtGFP) folds slowly and poorly in E. coli, thereby producing little fluorescence. These properties previously enabled directed evolution of chaperones in E. coli to improve chaperone activity and therefore increase wtGFP fluorescence brightness (Wang et al, 2002). Similarly, wtGFP fluorescence can be used to monitor the protein folding stress level in E. coli (Song et al, 2016). Of importance, the underlying reason why expressing proteins with GFP fusions in E. coli increase their overall folding levels is due to productive folding interactions between the GFP and known chaperones (Zhang et al, 2005). We have confirmed the previous findings that co-expressing known chaperones increases the fluorescence (and therefore the folded protein) of wtGFP in E. coli (Fig EV5). Click here to expand this figure. Figure EV5. The effect of nucleic acids on the fluorescence of wtGFP A. Expression vector constructs. Both vectors are under the control of pBAD promoter, which is induced by 0.2% L-Arabinose. B. Cellular fluorescence assay of wtGFP in the presence or absence of protein folding enhancing factors. White bars (+Empty IN and +Seq42) represent negative controls. The data are presented as mean ± SD (n = 3) of technical triplicate. C. In vitro refolding of wtGFP in the presence or absence of htDNA. GFP refolding curv
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