How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins.

Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self-avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N-terminal domain of pertactin (PNt), an IDP with an amino acid composition typical of folded proteins. While PNt contracts somewhat with temperature, it nevertheless remains expanded over 10-58°C, with a Flory exponent, ν, >0.50. Both low and high ionic strength also produce contraction in PNt, but this contraction is mitigated by reducing charge segregation. With 46% glycine and low hydrophobicity, the reduced form of snow flea anti-freeze protein (red-sfAFP) is unaffected by temperature and ionic strength and persists as a near-SARW, ν ~ 0.54, arguing that the thermal contraction of PNt is due to stronger interactions between hydrophobic side chains. Additionally, red-sfAFP is a proxy for the polypeptide backbone, which has been thought to collapse in water. Increasing the glycine segregation in red-sfAFP had minimal effect on ν. Water remained a good solvent even with 21 consecutive glycine residues (ν > 0.5), and red-sfAFP variants lacked stable backbone hydrogen bonds according to hydrogen exchange. Similarly, changing glycine segregation has little impact on ν in other glycine-rich proteins. These findings underscore the generality that many disordered states can be expanded and unstructured, and that the hydrophobic effect alone is insufficient to drive significant chain collapse for typical protein sequences.

HDX-MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker.

Eukaryotic cells form condensates to sense and adapt to their environment [S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Nat. Rev. Mol. Cell Biol. 18, 285-298 (2017), H. Yoo, C. Triandafillou, D. A. Drummond, J. Biol. Chem. 294, 7151-7159 (2019)]. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation [J. A. Riback et al., Cell 168, 1028-1040.e19 (2017)]. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen-deuterium exchange/mass spectrometry to investigate the mechanism of Pab1's condensation. Pab1's four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe populations allows us to identify which regions contribute to the condensate's interaction network. Our data yield a picture of Pab1's stress-triggered condensation, which we term sequential activation (Fig. 1A), wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.

Molecular insight into how the position of an abasic site modifies DNA duplex stability and dynamics.

Local perturbations to DNA base-pairing stability from lesions and chemical modifications can alter the stability and dynamics of an entire oligonucleotide. End effects may cause the position of a disruption within a short duplex to influence duplex stability and structural dynamics, yet this aspect of nucleic acid modifications is often overlooked. We investigate how the position of an abasic site (AP site) impacts the stability and dynamics of short DNA duplexes. Using a combination of steady-state and time-resolved spectroscopy and molecular dynamics simulations, we unravel an interplay between AP-site position and nucleobase sequence that controls energetic and dynamic disruption to the duplex. The duplex is disrupted into two segments by an entropic barrier for base-pairing on each side of the AP site. The barrier induces fraying of the short segment when an AP site is near the termini. Shifting the AP site inward promotes a transition from short-segment fraying to fully encompassing the barrier into the thermodynamics of hybridization, leading to further destabilization of the duplex. Nucleobase sequence determines the length scale for this transition by tuning the barrier height and base-pair stability of the short segment, and certain sequences enable out-of-register base-pairing to minimize the barrier height.

Molecular insight into how the position of an abasic site and its sequence environment influence DNA duplex stability and dynamics.

Local perturbations to DNA base-pairing stability from lesions and chemical modifications can alter the stability and dynamics of an entire oligonucleotide. End effects may cause the position of a disruption within a short duplex to influence duplex stability and structural dynamics, yet this aspect of nucleic acid modifications is often overlooked. We investigate how the position of an abasic site (AP site) impacts the stability and dynamics of short DNA duplexes. Using a combination of steady-state and time-resolved spectroscopy and molecular dynamics simulations, we unravel an interplay between AP-site position and nucleobase sequence that controls energetic and dynamic disruption to the duplex. The duplex is disrupted into two segments by an entropic barrier for base pairing on each side of the AP site. The barrier induces fraying of the short segment when an AP site is near the termini. Shifting the AP site inward promotes a transition from short-segment fraying to fully encompassing the barrier into the thermodynamics of hybridization, leading to further destabilization the duplex. Nucleobase sequence determines the length scale for this transition by tuning the barrier height and base-pair stability of the short segment, and certain sequences enable out-of-register base pairing to minimize the barrier height.

Thermodynamics and kinetics of DNA and RNA dinucleotide hybridization to gaps and overhangs.

Hybridization of short nucleic acid segments (<4 nt) to single-strand templates occurs as a critical intermediate in processes such as nonenzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood because of the experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential noncanonical base-pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2-40 µs depending on the template and temperature. Dinucleotide hybridization and dehybridization involve a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates.

Direct monitoring of the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization from gaps and overhangs.

Hybridization of short nucleic acid segments (<4 nucleotides) to single-strand templates occurs as a critical intermediate in processes such as non-enzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood due to experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential non-canonical base pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2 to 40 µs depending on the template and temperature. Dinucleotide hybridization and dehybridization involves a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Prediction and Validation of a Protein's Free Energy Surface Using Hydrogen Exchange and (Importantly) Its Denaturant Dependence.

The denaturant dependence of hydrogen-deuterium exchange (HDX) is a powerful measurement to identify the breaking of individual H-bonds and map the free energy surface (FES) of a protein including the very rare states. Molecular dynamics (MD) can identify each partial unfolding event with atomic-level resolution. Hence, their combination provides a great opportunity to test the accuracy of simulations and to verify the interpretation of HDX data. For this comparison, we use Upside, our new and extremely fast MD package that is capable of folding proteins with an accuracy comparable to that of all-atom methods. The FESs of two naturally occurring and two designed proteins are so generated and compared to our NMR/HDX data. We find that Upside's accuracy is considerably improved upon modifying the energy function using a new machine-learning procedure that trains for proper protein behavior including realistic denatured states in addition to stable native states. The resulting increase in cooperativity is critical for replicating the HDX data and protein stability, indicating that we have properly encoded the underlying physiochemical interactions into an MD package. We did observe some mismatch, however, underscoring the ongoing challenges faced by simulations in calculating accurate FESs. Nevertheless, our ensembles can identify the properties of the fluctuations that lead to HDX, whether they be small-, medium-, or large-scale openings, and can speak to the breadth of the native ensemble that has been a matter of debate.

Nanodroplet Oligomers (NanDOs) of Aß40.

Using atomic force microscopy (AFM) and nuclear magnetic resonance (NMR), we describe small Aß40 oligomers, termed nanodroplet oligomers (NanDOs), which form rapidly and at Aß40 concentrations too low for fibril formation. NanDOs were observed in putatively monomeric solutions of Aß40 (e.g., by size exclusion chromatography). Video-rate scanning AFM shows rapid fusion and dissolution of small oligomer-sized particles, of which the median size increases with peptide concentration. In NMR (13C HSQC), a small number of chemical shifts changed with a change in peptide concentration. Paramagnetic relaxation enhancement NMR experiments also support the formation of NanDOs and suggest prominent interactions in hydrophobic domains of Aß40. Addition of Zn2+ to Aß40 solutions caused flocculation of NanDO-containing solutions, and selective loss of signal intensity in NMR spectra from residues in the N-terminal domain of Aß40. NanDOs may represent the earliest aggregated form of Aß40 in the aggregation pathway and are akin to premicelles in solutions of amphiphilies.

Atomic-level differences between brain parenchymal- and cerebrovascular-seeded Aß fibrils.

Alzheimer's disease is characterized by neuritic plaques, the main protein components of which are ß-amyloid (Aß) peptides deposited as ß-sheet-rich amyloid fibrils. Cerebral Amyloid Angiopathy (CAA) consists of cerebrovascular deposits of Aß peptides; it usually accompanies Alzheimer's disease, though it sometimes occurs in the absence of neuritic plaques, as AD also occurs without accompanying CAA. Although neuritic plaques and vascular deposits have similar protein compositions, one of the characteristic features of amyloids is polymorphism, i.e., the ability of a single pure peptide to adopt multiple conformations in fibrils, depending on fibrillization conditions. For this reason, we asked whether the Aß fibrils in neuritic plaques differed structurally from those in cerebral blood vessels. To address this question, we used seeding techniques, starting with amyloid-enriched material from either brain parenchyma or cerebral blood vessels (using meninges as the source). These amyloid-enriched preparations were then added to fresh, disaggregated solutions of Aß to make replicate fibrils, as described elsewhere. Such fibrils were then studied by solid-state NMR, fiber X-ray diffraction, and other biophysical techniques. We observed chemical shift differences between parenchymal vs. vascular-seeded replicate fibrils in select sites (in particular, Ala2, Phe4, Val12, and Gln15 side chains) in two-dimensional 13C-13C correlation solid-state NMR spectra, strongly indicating structural differences at these sites. X-ray diffraction studies also indicated that vascular-seeded fibrils displayed greater order than parenchyma-seeded fibrils in the "side-chain dimension" (~ 10 Å reflection), though the "hydrogen-bond dimensions" (~ 5 Å reflection) were alike. These results indicate that the different nucleation conditions at two sites in the brain, parenchyma and blood vessels, affect the fibril products that get formed at each site, possibly leading to distinct pathophysiological outcomes.

ß-amyloid model core peptides: Effects of hydrophobes and disulfides.

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of ß-amyloid (Aß21-30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aß, and variants of these either cyclized or with an N-terminal Cys. While Aß21-30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aß16-34 led to formation of typical amyloid fibrils. NMR showed no long-range nuclear overhauser effect (nOes) in Aß21-30, Aß16-34, or their variants, however. Serial 1 H-15 N-heteronuclear single quantum coherence spectroscopy, 1 H-1 H nuclear overhauser effect spectroscopy, and 1 H-1 H total correlational spectroscopy spectra were used to follow aggregation of Aß16-34 and Cys-Aß16-34 at a site-specific level. The addition of an N-terminal Cys residue (in Cys-Aß16-34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aß16-34 and Cys-Aß16-34, according to which Cys-Aß16-34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.

ß-Amyloid aggregation and heterogeneous nucleation.

In this article, we consider the role of heterogeneous nucleation in ß-amyloid aggregation. Heterogeneous nucleation is more common and occurs at lower levels of supersaturation than homogeneous nucleation. The nucleation period is also the stage at which most of the polymorphism of amyloids arises, this being one of the defining features of amyloids. We focus on several well-known heterogeneous nucleators of ß-amyloid, including lipid surfaces, especially those enriched in gangliosides and cholesterol, and divalent metal ions. These two broad classes of nucleators affect ß-amyloid particularly in light of the amphiphilicity of these peptides: the N-terminal region, which is largely polar and charged, contains the metal binding site, whereas the C-terminal region is aliphatic and is important in lipid binding. Notably, these two classes of nucleators can interact cooperatively, aggregation begetting greater aggregation.

A versatile method for producing labeled or unlabeled Aß55, Aß40, and other ß-amyloid family peptides.

We present a straightforward, versatile method for expressing and purifying ß-amyloid (Aß40) and transmembrane peptides derived from ß-amyloid precursor protein (Aß55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag with a flexible, hydrophilic linker sequence, followed by a cleavage site, and then Aß peptides. The HexaHis tag rather than a protein fusion partner (e.g., GST) obviates the need for a folded protein in affinity purification. Second, we present two cleavage methods, using either Factor Xa or BNPS-Skatole. Although the latter procedure requires subsequent reduction of the product, we describe methods for minimizing side reactions. Because the use of BNPS-Skatole obviates the need for a folded protein in the cleavage reaction, it is compatible with harsh conditions (e.g., inclusion of detergents and denaturants) needed to solubilize the fusion proteins; such conditions tend to inactivate Factor Xa. Finally, we also describe purification strategies for Aß40 and Aß55 using FPLC and/or reverse phase HPLC. Yields of peptide after these BNPS-Skatole cleavage and peptide reduction, though subquantitative, greatly exceed those obtained using Factor Xa cleavage, as the reaction of BNPS-Skatole is insensitive to the presence of detergents and denaturants, and therefore can be used to produce highly aggregative and low solubility peptides such as Aß55. Trp is a low abundance amino acid in proteins generally, and for peptides like Aß55, and other transmembane peptides lacking Trp in relevant positions, this cleavage method remains a useful option.

Magnetic resonance spectroscopy detects differential lipid composition in mammary glands on low fat, high animal fat versus high fructose diets.

The effects of consumption of different diets on the fatty acid composition in the mammary glands of SV40 T-antigen (Tag) transgenic mice, a well-established model of human triple-negative breast cancer, were investigated with magnetic resonance spectroscopy and spectroscopic imaging. Female C3(1) SV40 Tag transgenic mice (n = 12) were divided into three groups at 4 weeks of age: low fat diet (LFD), high animal fat diet (HAFD), and high fructose diet (HFruD). MRI scans of mammary glands were acquired with a 9.4 T scanner after 8 weeks on the diet. 1H spectra were acquired using point resolved spectroscopy (PRESS) from two 1 mm3 boxes on each side of inguinal mammary gland with no cancers, lymph nodes, or lymph ducts. High spectral and spatial resolution (HiSS) images were also acquired from nine 1-mm slices. A combination of Gaussian and Lorentzian functions was used to fit the spectra. The percentages of poly-unsaturated fatty acids (PUFA), mono-unsaturated fatty acids (MUFA), and saturated fatty acids (SFA) were calculated from each fitted spectrum. Water and fat peak height images (maps) were generated from HiSS data. The results showed that HAFD mice had significantly lower PUFA than both LFD (p < 0.001) and HFruD (p < 0.01) mice. The mammary lipid quantity calculated from 1H spectra was much larger in HAFD mice than in LFD (p = 0.03) but similar to HFruD mice (p = 0.10). The average fat signal intensity over the mammary glands calculated from HiSS fat maps was ~60% higher in HAFD mice than in LFD (p = 0.04) mice. The mean or median of calculated parameters for the HFruD mice were between those for LFD and HAFD mice. Therefore, PRESS spectroscopy and HiSS MRI demonstrated water and fat composition changes in mammary glands due to a Western diet, which was low in potassium, high in sodium, animal fat, and simple carbohydrates. Measurements of PUFA with MRI could be used to evaluate cancer risk, improve cancer detection and diagnosis, and guide preventative therapy.

Metabolism of megestrol acetate in vitro and the role of oxidative metabolites.

1. There is limited knowledge regarding the metabolism of megestrol acetate (MA), as it was approved by FDA in 1971, prior to the availability of modern tools for identifying specific drug-metabolizing enzymes. We determined the cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) that metabolize MA, identified oxidative metabolites and determined pharmacologic activity at the progesterone, androgen and glucocorticoid receptors (PR, AR and GR, respectively). 2. Oxidative metabolites were produced using human liver microsomes (HLMs), and isolated for mass spectral (MS) and nuclear magnetic resonance (NMR) analyses. We screened recombinant P450s using MA at 62 µM (HLM Km for metabolite 1; M1) and 28 µM (HLM Km for metabolite 2; M2). UGT isoforms were simultaneously incubated with UDPGA, nicotinamide adenine dinucleotide phosphate (NADPH), CYP3A4 and MA. Metabolites were evaluated for pharmacologic activity on the PR, AR and GR. CYP3A4 and CYP3A5 are responsible for oxidative metabolism of 62 µM MA. 3. At 28 µM substrate concentration, CYP3A4 was the only contributing enzyme. Mass spectral and NMR data suggest metabolism of MA to two alcohols. After oxidation, MA is converted into two secondary glucuronides by UGT2B17 among other UGTs. MA, M1 and M2 had significant pharmacologic activity on the PR while only MA showed activity on the AR and GR.

Allosteric control of a bacterial stress response system by an anti-σ factor.

Bacterial signal transduction systems commonly use receiver (REC) domains, which regulate adaptive responses to the environment as a function of their phosphorylation state. REC domains control cell physiology through diverse mechanisms, many of which remain understudied. We have defined structural features that underlie activation of the multi-domain REC protein, PhyR, which functions as an anti-anti-σ factor and regulates transcription of genes required for stress adaptation and host-microbe interactions in Alphaproteobacteria. Though REC phosphorylation is necessary for PhyR function in vivo, we did not detect expected changes in inter-domain interactions upon phosphorylation by solution X-ray scattering. We sought to understand this result by defining additional molecular requirements for PhyR activation. We uncovered specific interactions between unphosphorylated PhyR and an intrinsically disordered region (IDR) of the anti-σ factor, NepR, by solution NMR spectroscopy. Our data support a model whereby nascent NepR(IDR)-PhyR interactions and REC phosphorylation coordinately impart the free energy to shift PhyR to an open, active conformation that binds and inhibits NepR. This mechanism ensures PhyR is activated only when NepR and an activating phosphoryl signal are present. Our study provides new structural understanding of the molecular regulatory logic underlying a conserved environmental response system.

Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vγ9Vδ2 T cell activation.

Human Vγ9Vδ2 T cells respond to microbial infections as well as certain types of tumors. The key initiators of Vγ9Vδ2 activation are small, pyrophosphate-containing molecules called phosphoantigens (pAgs) that are present in infected cells or accumulate intracellularly in certain tumor cells. Recent studies demonstrate that initiation of the Vγ9Vδ2 T cell response begins with sensing of pAg via the intracellular domain of the butyrophilin 3A1 (BTN3A1) molecule. However, it is unknown how downstream events can ultimately lead to T cell activation. Here, using NMR spectrometry and molecular dynamics (MD) simulations, we characterize a global conformational change in the B30.2 intracellular domain of BTN3A1 induced by pAg binding. We also reveal by crystallography two distinct dimer interfaces in the BTN3A1 full-length intracellular domain, which are stable in MD simulations. These interfaces lie in close proximity to the pAg-binding pocket and contain clusters of residues that experience major changes of chemical environment upon pAg binding. This suggests that pAg binding disrupts a preexisting conformation of the BTN3A1 intracellular domain. Using a combination of biochemical, structural, and cellular approaches we demonstrate that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminishes pAg-induced T cell activation. Based on these results, we propose a model in which a conformational change in BTN3A1 is a key event of pAg sensing that ultimately leads to T cell activation.

Aromatic claw: A new fold with high aromatic content that evades structural prediction.

We determined the NMR structure of a highly aromatic (13%) protein of unknown function, Aq1974 from Aquifex aeolicus (PDB ID: 5SYQ). The unusual sequence of this protein has a tryptophan content five times the normal (six tryptophan residues of 114 or 5.2% while the average tryptophan content is 1.0%) with the tryptophans occurring in a WXW motif. It has no detectable sequence homology with known protein structures. Although its NMR spectrum suggested that the protein was rich in ß-sheet, upon resonance assignment and solution structure determination, the protein was found to be primarily α-helical with a small two-stranded ß-sheet with a novel fold that we have termed an Aromatic Claw. As this fold was previously unknown and the sequence unique, we submitted the sequence to CASP10 as a target for blind structural prediction. At the end of the competition, the sequence was classified a hard template based model; the structural relationship between the template and the experimental structure was small and the predictions all failed to predict the structure. CSRosetta was found to predict the secondary structure and its packing; however, it was found that there was little correlation between CSRosetta score and the RMSD between the CSRosetta structure and the NMR determined one. This work demonstrates that even in relatively small proteins, we do not yet have the capacity to accurately predict the fold for all primary sequences. The experimental discovery of new folds helps guide the improvement of structural prediction methods.

N(6)-Methyladenosine Modification in a Long Noncoding RNA Hairpin Predisposes Its Conformation to Protein Binding.

N(6)-Methyladenosine (m(6)A) is a reversible and abundant internal modification of messenger RNA (mRNA) and long noncoding RNA (lncRNA) with roles in RNA processing, transport, and stability. Although m(6)A does not preclude Watson-Crick base pairing, the N(6)-methyl group alters the stability of RNA secondary structure. Since changes in RNA structure can affect diverse cellular processes, the influence of m(6)A on mRNA and lncRNA structure has the potential to be an important mechanism for m(6)A function in the cell. Indeed, an m(6)A site in the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was recently shown to induce a local change in structure that increases the accessibility of a U5-tract for recognition and binding by heterogeneous nuclear ribonucleoprotein C (HNRNPC). This m(6)A-dependent regulation of protein binding through a change in RNA structure, termed "m(6)A-switch", affects transcriptome-wide mRNA abundance and alternative splicing. To further characterize this first example of an m(6)A-switch in a cellular RNA, we used nuclear magnetic resonance and Förster resonance energy transfer to demonstrate the effect of m(6)A on a 32-nucleotide RNA hairpin derived from the m(6)A-switch in MALAT1. The observed imino proton nuclear magnetic resonance resonances and Förster resonance energy transfer efficiencies suggest that m(6)A selectively destabilizes the portion of the hairpin stem where the U5-tract is located, increasing the solvent accessibility of the neighboring bases while maintaining the overall hairpin structure. The m(6)A-modified hairpin has a predisposed conformation that resembles the hairpin conformation in the RNA-HNRNPC complex more closely than the unmodified hairpin. The m(6)A-induced structural changes in the MALAT1 hairpin can serve as a model for a large family of m(6)A-switches that mediate the influence of m(6)A on cellular processes.

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis­two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction­caused by defects in mitophagy­can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.