Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.
- MeSH
- malá interferující RNA MeSH
- messenger RNA genetika MeSH
- mozek MeSH
- myši MeSH
- nanočástice * MeSH
- nemoci nervového systému * MeSH
- vodící RNA, systémy CRISPR-Cas MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
An increase in phosphorylation of the Tau protein is associated with Alzheimer's disease (AD) progression through unclear molecular mechanisms. In general, phosphorylation modifies the interaction of intrinsically disordered proteins, such as Tau, with other proteins; however, elucidating the structural basis of this regulation mechanism remains challenging. The bridging integrator-1 gene is an AD genetic determinant whose gene product, BIN1, directly interacts with Tau. The proline-rich motif recognized within a Tau(210-240) peptide by the SH3 domain of BIN1 (BIN1 SH3) is defined as 216PTPP219, and this interaction is modulated by phosphorylation. Phosphorylation of T217 within the Tau(210-240) peptide led to a 6-fold reduction in the affinity, while single phosphorylation at either T212, T231, or S235 had no effect on the interaction. Nonetheless, combined phosphorylation of T231 and S235 led to a 3-fold reduction in the affinity, although these phosphorylations are not within the BIN1 SH3-bound region of the Tau peptide. Using nuclear magnetic resonance (NMR) spectroscopy, these phosphorylations were shown to affect the local secondary structure and dynamics of the Tau(210-240) peptide. Models of the (un)phosphorylated peptides were obtained from molecular dynamics (MD) simulation validated by experimental data and showed compaction of the phosphorylated peptide due to increased salt bridge formation. This dynamic folding might indirectly impact the BIN1 SH3 binding by a decreased accessibility of the binding site. Regulation of the binding might thus not only be due to local electrostatic or steric effects from phosphorylation but also to the modification of the conformational properties of Tau.
- MeSH
- adaptorové proteiny signální transdukční metabolismus MeSH
- Alzheimerova nemoc * metabolismus MeSH
- fosforylace MeSH
- jaderné proteiny metabolismus MeSH
- lidé MeSH
- nádorové supresorové proteiny chemie MeSH
- peptidy chemie MeSH
- prolin metabolismus MeSH
- proteiny tau * metabolismus MeSH
- src homologní domény MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.
- MeSH
- Bacillus thuringiensis enzymologie ultrastruktura MeSH
- cytokiny chemie genetika MeSH
- Escherichia coli enzymologie ultrastruktura MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- leukocyty mononukleární chemie enzymologie MeSH
- lidé MeSH
- membránové proteiny chemie genetika ultrastruktura MeSH
- nukleotidy biosyntéza chemie genetika MeSH
- přirozená imunita genetika MeSH
- substrátová specifita MeSH
- Thermotoga maritima enzymologie ultrastruktura MeSH
- Vibrio cholerae enzymologie ultrastruktura MeSH
- vztahy mezi strukturou a aktivitou * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.
- MeSH
- bodová mutace * MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- membránové proteiny chemie genetika metabolismus MeSH
- molekulární struktura MeSH
- nukleotidy cyklické chemie metabolismus MeSH
- proteinové domény MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lipid transfer from lipoprotein particles to cells is essential for lipid homeostasis. High-density lipoprotein (HDL) particles are mainly captured by cell membrane-associated scavenger receptor class B type 1 (SR-B1) from the bloodstream, while low-density and very-low-density lipoprotein (LDL and VLDL, respectively) particles are mostly taken up by receptor-mediated endocytosis. However, the role of the target lipid membrane itself in the transfer process has been largely neglected so far. Here, we study how lipoprotein particles (HDL, LDL, and VLDL) interact with synthetic lipid bilayers and cell-derived membranes and transfer their cargo subsequently. Employing cryo-electron microscopy, spectral imaging, and fluorescence (cross) correlation spectroscopy allowed us to observe integration of all major types of lipoprotein particles into the membrane and delivery of their cargo in a receptor-independent manner. Importantly, the biophysical properties of the target cell membranes change upon delivery of cargo. The concept of receptor-independent interaction of lipoprotein particles with membranes helps us to better understand lipoprotein particle biology and can be exploited for novel treatments of dyslipidemia diseases.
Staphylococcus aureus uses IsdG and IsdI to convert heme into a mixture of staphylobilin isomers, 15-oxo-β-bilirubin and 5-oxo-δ-bilirubin, formaldehyde, and iron. The highly ruffled heme found in the heme-IsdI and IsdG complexes has been proposed to be responsible for the unique heme degradation products. We employed resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopies to examine the coordination and electronic structures of heme bound to IsdG and IsdI. Heme complexed to IsdG and IsdI is coordinated by a neutral histidine. The trans ligand is hydroxide in the ferric alkaline form of both proteins. In the ferric neutral form at pH 6.0, heme is six-coordinated with water as the sixth ligand for IsdG and is in the mixture of the five-coordinated and six-coordinated species for IsdI. In the ferrous CO-bound form, CO is strongly hydrogen bonded with a distal residue. The marker lines, ν2 and ν3, appear at frequencies that are distinct from other proteins having planar hemes. The EPR spectra for the ferric hydroxide and cyanide states might be explained by assuming the thermal mixing of the d-electron configurations, (dxy)2(dxz,dyz)3 and (dxz,dyz)4(dxy)1. The fraction for the latter becomes larger for the ferric cyanide form. In the ferric neutral state at pH 6.0, the quantum mechanical mixing of the high and intermediate spin configurations might explain the peculiar frequencies of ν2 and ν3 in the RR spectra. The heme ruffling imposed by IsdG and IsdI gives rise to unique electronic structures of heme, which are expected to modulate the first and subsequent steps of the heme oxygenation.
- MeSH
- bakteriální proteiny chemie MeSH
- elektronová paramagnetická rezonance MeSH
- hem chemie MeSH
- lidé MeSH
- oxid uhelnatý chemie MeSH
- oxygenasy se smíšenou funkcí chemie MeSH
- oxygenasy chemie MeSH
- Ramanova spektroskopie MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus chemie MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human cyclophilin D is a mitochondrial peptidyl-prolyl isomerase that plays a role in regulating the opening of the mitochondrial permeability transition pore. It is considered a viable and promising molecular target for the treatment of diseases for which disease development is associated with pore opening, e.g., Alzheimer's disease or ischemia/reperfusion injury. Currently available and widely used in vitro methods based on Kofron's assay for determining cyclophilin D activity suffer from serious drawbacks and limitations. In this study, a completely novel approach for an in vitro assay of cyclophilin D activity using RNase T1 refolding is introduced. The method is simple and is more in line with the presumed physiological role of cyclophilin D in protein folding than Kofron's assay, which relies on a peptide substrate. The method is applicable for identifying novel inhibitors of cyclophilin D as potential drugs for the treatment of the diseases mentioned above. Moreover, the description of CypD activity in the in vitro RNase T1 refolding assay reveals new possibilities for investigating the role of cyclophilin D in protein folding in cells and may lead to a better understanding of its pathological and physiological roles.
- MeSH
- Aspergillus oryzae enzymologie MeSH
- guanyloribonukleasa chemie MeSH
- konformace proteinů MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- molekulární modely MeSH
- objevování léků * MeSH
- peptidylprolylisomerasa F chemie metabolismus MeSH
- refolding proteinů * MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
We developed a one-step direct assay for the determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety with sulfur, resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiencies ranging from not accepted for HDAC1 to kinetic constants similar to that of the parent oxo substrate for HDAC8. Class II HDACs can hydrolyze thioacylated substrates with approximately 5-10-fold reduced kcat values, which resembles the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficiently with an ∼5-fold reduced specificity constant. On the basis of the unique spectroscopic properties of thioamide bonds (strong absorption in spectral range of 260-280 nm and efficient fluorescence quenching), HDAC-mediated cleavage of thioamides could be followed by ultraviolet-visible and fluorescence spectroscopy in a continuous manner. The HDAC activity assay is compatible with microtiter plate-based screening formats up to 1536-well plates with Z' factors of >0.75 and signal-to-noise ratios of >50. Using thioacylated lysine residues in p53-derived peptides, we optimized substrates for HDAC8 with a catalytic efficiency of >250000 M-1 s-1, which are more than 100-fold more effective than most of the known substrates. We determined inhibition constants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values from the literature. On the other hand, we could introduce N-methylated, N-acylated lysine residues as inhibitors for HDACs with an IC50 value of 1 μM for an N-methylated, N-myristoylated peptide derivative and human HDAC11.
- MeSH
- biokatalýza MeSH
- histondeacetylasy chemie genetika metabolismus MeSH
- inhibitory histondeacetylas chemie metabolismus MeSH
- kinetika MeSH
- lidé MeSH
- lysin chemie metabolismus MeSH
- thioamidy chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
G-Quadruplexes are four-stranded nucleic acid structures typically stabilized by GGGG tetrads. These structures are intrinsically fluorescent, which expands the known scope of nucleic acid function and raises the possibility that they could eventually be used as signaling components in label-free sensors constructed from DNA or RNA. In this study, we systematically investigated the effects of mutations in tetrads, loops, and overhanging nucleotides on the fluorescence intensity and maximum emission wavelength of >500 sequence variants of a reference DNA G-quadruplex. Some of these mutations modestly increased the fluorescence intensity of this G-quadruplex, while others shifted its maximum emission wavelength. Mutations that increased the fluorescence intensity were distinct from those that increased the maximum emission wavelength, suggesting a trade-off between these two biochemical properties. The fluorescence intensity and maximum emission wavelength were also correlated with multimeric state: the most fluorescent G-quadruplexes were monomers, while those with the highest maximum emission wavelengths typically formed dimeric structures. Oligonucleotides containing multiple G-quadruplexes were in some cases more fluorescent than those containing a single G-quadruplex, although this depended on the length and sequence of the spacer linking the G-quadruplexes. These experiments provide new insights into the properties of fluorescent G-quadruplexes and should aid in the development of improved label-free nucleic acid sensors.
Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2', which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications.
- MeSH
- fosforylace MeSH
- insulinu podobný růstový faktor I chemie genetika MeSH
- insulinu podobný růstový faktor II chemie genetika MeSH
- inzulin chemie metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- molekulární evoluce MeSH
- mutace MeSH
- protein - isoformy MeSH
- receptor inzulinu chemie metabolismus MeSH
- receptory somatomedinů chemie genetika MeSH
- signální transdukce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
