Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.

A dietary anthocyanin cyanidin-3-O-glucoside binds to PPARs to regulate glucose metabolism and insulin sensitivity in mice.

We demonstrate the mechanism by which C3G, a major dietary anthocyanin, regulates energy metabolism and insulin sensitivity. Oral administration of C3G reduced hepatic and plasma triglyceride levels, adiposity, and improved glucose tolerance in mice fed high-fat diet. Hepatic metabolomic analysis revealed that C3G shifted metabolite profiles towards fatty acid oxidation and ketogenesis. C3G increased glucose uptake in HepG2 cells and C2C12 myotubes and induced the rate of hepatic fatty acid oxidation. C3G directly interacted with and activated PPARs, with the highest affinity for PPARα. The ability of C3G to reduce plasma and hepatic triglycerides, glucose tolerance, and adiposity and to induce oxygen consumption and energy expenditure was abrogated in PPARα-deficient mice, suggesting that PPARα is the major target for C3G. These findings demonstrate that the dietary anthocyanin C3G activates PPARs, a master regulators of energy metabolism. C3G is an agonistic ligand of PPARs and stimulates fuel preference to fat.

Azelaic Acid Induces Mitochondrial Biogenesis in Skeletal Muscle by Activation of Olfactory Receptor 544.

Mouse olfactory receptor 544 (Olfr544) is ectopically expressed in varied extra-nasal organs with tissue specific functions. Here, we investigated the functionality of Olfr544 in skeletal muscle cells and tissue. The expression of Olfr544 is confirmed by RT-PCR and qPCR in skeletal muscle cells and mouse skeletal muscle assessed by RT-PCR and qPCR. Olfr544 activation by its ligand, azelaic acid (AzA, 50 µM), induced mitochondrial biogenesis and autophagy in cultured skeletal myotubes by induction of cyclic adenosine monophosphate-response element binding protein (CREB)-peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-extracellular signal-regulated kinase-1/2 (ERK1/2) signaling axis. The silencing Olfr544 gene expression abrogated these effects of AzA in cultured myotubes. Similarly, in mice, the acute subcutaneous injection of AzA induced the CREB-PGC-1α-ERK1/2 pathways in mouse skeletal muscle, but these activations were negated in those of Olfr544 knockout mice. These demonstrate that the induction of mitochondrial biogenesis in skeletal muscle by AzA is Olfr544-dependent. Oral administration of AzA to high-fat-diet fed obese mice for 6 weeks increased mitochondrial DNA content in the skeletal muscle as well. Collectively, these findings demonstrate that Olfr544 activation by AzA regulates mitochondrial biogenesis in skeletal muscle. Intake of AzA or food containing AzA may help to improve skeletal muscle function.

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.

Olfactory receptor 43 reduces hepatic lipid accumulation and adiposity in mice.

Olfactory receptors are primarily expressed in nasal olfactory epithelium, but these receptors are also ectopically expressed in diverse tissues. In this study, we investigated the biological functions of Olfr43, a mouse homolog of human OR1A1, in cultured hepatocytes and mice to assess its functionality in lipid metabolism. Olfr43 was expressed in mouse hepatocytes, and Olfr43 activation by a known ligand, (-)-carvone, stimulated cAMP response element-binding protein (CREB) activity. In ligand-receptor binding studies using site-directed mutagenesis, (-)-carvone binding required two residues, M257 and Y258, in Olfr43. In the mouse study, oral administration of (-)-carvone for 5 weeks in high-fat diet-fed mice improved energy metabolism, including reductions in hepatic steatosis and adiposity, and improved glucose and insulin tolerance. In mouse livers and cultured mouse hepatocytes, Olfr43 activation simulated the CREB-hairy and enhancer of split 1 (HES1)-peroxisome proliferator-activated receptor (PPAR)-γ signaling axis, leading to a reduction in hepatic triglyceride accumulation in the mouse liver. Thus, long-term administration of (-)-carvone reduces hepatic steatosis. The knockdown of Olfr43 gene expression in cultured hepatocytes negated these effects of (-)-carvone. In conclusion, an ectopic olfactory receptor, hepatic Olfr43, regulates energy metabolism via the CREB-HES1-PPARγ signaling axis.

Adenylate kinase potentiates the capsular polysaccharide by modulating Cps2D in Streptococcus pneumoniae D39.

Streptococcus pneumoniae is a polysaccharide-encapsulated bacterium. The capsule thickens during blood invasion compared with the thinner capsules observed in asymptomatic nasopharyngeal colonization. However, the underlying mechanism regulating differential CPS expression remains unclear. CPS synthesis requires energy that is supplied by ATP. Previously, we demonstrated a correlation between ATP levels and adenylate kinase in S. pneumoniae (SpAdK). A dose-dependent induction of SpAdK in serum was also reported. To meet medical needs, this study aimed to elucidate the role of SpAdK in the regulation of CPS production. CPS levels in S. pneumoniae type 2 (D39) increased proportionally with SpAdK levels, but they were not related to pneumococcal autolysis. Moreover, increased SpAdK levels resulted in increased total tyrosine kinase Cps2D levels and phosphorylated Cps2D, which is a regulator of CPS synthesis in the D39 strain. Our results also indicated that the SpAdK and Cps2D proteins interact in the presence of Mg-ATP. In addition, in silico analysis uncovered the mechanism behind this protein-protein interaction, suggesting that SpAdK binds with the Cps2D dimer. This established the importance of the ATP-binding domain of Cps2D. Taken together, the biophysical interaction between SpAdK and Cps2D plays an important role in enhancing Cps2D phosphorylation, which results in increased CPS synthesis.

Peptide-Programmable Nanoparticle Superstructures with Tailored Electrocatalytic Activity.

Biomaterials derived via programmable supramolecular protein assembly provide a viable means of constructing precisely defined structures. Here, we present programmed superstructures of AuPt nanoparticles (NPs) on carbon nanotubes (CNTs) that exhibit distinct electrocatalytic activities with respect to the nanoparticle positions via rationally modulated peptide-mediated assembly. De novo designed peptides assemble into six-helix bundles along the CNT axis to form a suprahelical structure. Surface cysteine residues of the peptides create AuPt-specific nucleation site, which allow for precise positioning of NPs onto helical geometries, as confirmed by 3-D reconstruction using electron tomography. The electrocatalytic model system, i.e., AuPt for oxygen reduction, yields electrochemical response signals that reflect the controlled arrangement of NPs in the intended assemblies. Our design approach can be expanded to versatile fields to build sophisticated functional assemblies.

Hexacosanol reduces plasma and hepatic cholesterol by activation of AMP-activated protein kinase and suppression of sterol regulatory element-binding protein-2 in HepG2 and C57BL/6J mice.

Policosanols have hypocholesterolemic activity; however, the molecular mechanism of the policosanol effects is currently poorly characterized. We hypothesized that hexacosanol, a policosanol compound derived from barley sprout, may decrease cellular and plasma cholesterol levels; we thus investigated the hypocholesterolemic activity and mechanism of hexacosanol on both hepatocytes and high-fat-induced obese C57BL/6J mice. The reduction of total cholesterol, free cholesterol, and cholesteryl ester concentrations was confirmed in hexacosanol-stimulated hepatocytes (-38%, -33%, and -53%, respectively). Plasma, hepatic cholesterol concentrations, and hepatic steatosis were significantly reduced in high-fat-fed mice orally administered with hexacosanol (0.7 mg/kg body weight a day) for 8 weeks compared with those of vehicle-fed control mice (-15% and -40%, respectively). Hexacosanol in fact bound to the allosteric regulation site of AMP-activated protein kinase (AMPK)-ß subunit and thus activated AMPK that inhibited the activity of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase by inhibitory phosphorylation. In addition, activation of AMPK by hexacosanol induced hepatic autophagy activity, which could further reduce hepatic lipid accumulation. Alternatively, hexacosanol suppressed the nuclear translocation and activation of sterol regulatory element-binding protein-2 (SREBP-2), a key transcription factor in cholesterol biosynthesis. These results collectively suggest that hexacosanol is a major hypocholesterolemic compound in barley sprouts with regulation of AMPK activation and SREBP-2 suppression. These suppress 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase at both mRNA expression and protein activity levels. In conclusion, hexacosanol activates AMPK and hepatic autophagy and inhibits SREBP2, resulting in hypocholesterolemic activities and improvement of hepatic steatosis.

Molecular determinants of the olfactory receptor Olfr544 activation by azelaic acid.

The mouse olfactory receptor Olfr544 is expressed in several non-olfactory tissues and has been suggested as a functional receptor regulating different signaling pathways. However, the molecular interaction between Olfr544 and its natural ligand, azelaic acid (AzA), remains poorly characterized, primarily due to difficulties in the heterologous expression of the receptor protein on the cell membrane and lack of entire protein structure. In this report, we describe the molecular determinants of Olfr544 activation by AzA. N-terminal lucy-flag-rho tag ensured the heterologous expression of Olfr544 on the Hana3A cell surface. Molecular modeling and docking combined with mutational analysis identified amino acid residues in the Olfr544 for the interaction with AzA. Our data demonstrated that the Y109 residue in transmembrane helix 3 forms a hydrogen bond with AzA, which is crucial for the receptor-ligand interaction and activation. Y109 is required for the Olfr544 activation by AzA which, in turn, stimulates the Olfr544-dependent CREB-PGC-1α signaling axis and is followed by the induction of mitochondrial biogenesis in Olfr544 wild-type transfected Hana3A cells, but not in mock or Y109A mutant transfected cells. Collectively, these data indicated that a hydrogen bond between Y109 residue and AzA is a major determinant of the Olfr544-AzA interaction and activation.

Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes.

Long-term topical skin care by traditional anti-melanogenic agents can raise several safety concerns. An understanding of the molecular mechanisms of active compounds on melanogenesis is, therefore, necessary to address pigmentation issues. Here we revealed that stimulation with 1 mM betaine, an abundant component in rice bran, significantly reduced 21% of intracellular melanin content by suppressing tyrosinase activity and microphthalmia-associated transcription factor (MITF) expression in B16-F1 murine melanocytes. The expression of MITF was suppressed at both mRNA and protein levels by 43 and 44%, respectively. Subsequently, the betaine-stimulated melanocytes showed inhibition of PKA-CREB signaling axis but activation of extracellular-signal-regulated kinase and AKT-GSK3ß signaling pathways. This inhibition and activation led to downregulation of MITF expression at both the transcriptional and post-translational levels to suppress melanin synthesis. These findings collectively suggested that betaine is a potential anti-melanogenic compound for functional foods and cosmetics.

Syringaresinol induces mitochondrial biogenesis through activation of PPARß pathway in skeletal muscle cells.

Activation of peroxisome proliferator-activated receptors (PPARs) plays a crucial role in cellular energy metabolism that directly impacts mitochondrial biogenesis. In this study, we demonstrate that syringaresinol, a pharmacological lignan extracted from Panax ginseng berry, moderately binds to and activates PPARß with KD and EC50 values of 27.62±15.76µM and 18.11±4.77µM, respectively. Subsequently, the expression of peroxisome proliferator-activated receptor γ coactivator-1α together with PPARß transcriptional targets, mitochondrial carnitine palmitoyltransferase 1 and uncoupling protein 2, was also enhanced in terms of both mRNA and protein levels. The activation of these proteins induced mitochondrial biogenesis by enrichment of mitochondrial replication and density within C2C12 myotubes. Importantly, knockdown of PPARß reduced the syringaresinol-induced protein expression followed by the significant reduction of mitochondrial biogenesis. Taken together, our results indicate that syringaresinol induces mitochondrial biogenesis by activating PPARß pathway.

New conformations of linear polyubiquitin chains from crystallographic and solution-scattering studies expand the conformational space of polyubiquitin.

The conformational flexibility of linkage-specific polyubiquitin chains enables ubiquitylated proteins and their receptors to be involved in a variety of cellular processes. Linear or Met1-linked polyubiquitin chains, associated with nondegradational cellular signalling pathways, have been known to adopt multiple conformations from compact to extended conformations. However, the extent of such conformational flexibility remains open. Here, the crystal structure of linear Ub2 was determined in a more compact conformation than that of the previously known structure (PDB entry 3axc). The two structures differ significantly from each other, as shown by an r.m.s.d. between C(α) atoms of 3.1 Å. The compactness of the linear Ub2 structure in comparison with PDB entry 3axc is supported by smaller values of the radius of gyration (Rg; 18 versus 18.9 Å) and the maximum interatomic distance (Dmax; 55.5 versus 57.8 Å). Extra intramolecular hydrogen bonds formed among polar residues between the distal and proximal ubiquitin moieties seem to contribute to stabilization of the compact conformation of linear Ub2. An ensemble of three semi-extended and extended conformations of linear Ub2 was also observed by small-angle X-ray scattering (SAXS) analysis in solution. In addition, the conformational heterogeneity in linear polyubiquitin chains is clearly manifested by SAXS analyses of linear Ub3 and Ub4: at least three distinct solution conformations are observed in each chain, with the linear Ub3 conformations being compact. The results expand the extent of conformational space of linear polyubiquitin chains and suggest that changes in the conformational ensemble may be pivotal in mediating multiple signalling pathways.

Molecular determinants of polyubiquitin recognition by continuous ubiquitin-binding domains of Rad18.

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub2-Ub4) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the "extended LR motif" (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub2, not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.

New crystal structures of adenylate kinase from Streptococcus pneumoniae D39 in two conformations.

Adenylate kinases (AdKs; EC 2.7.3.4) play a critical role in intercellular homeostasis by the interconversion of ATP and AMP to two ADP molecules. Crystal structures of adenylate kinase from Streptococcus pneumoniae D39 (SpAdK) have recently been determined using ligand-free and inhibitor-bound crystals belonging to space groups P21 and P1, respectively. Here, new crystal structures of SpAdK in ligand-free and inhibitor-bound states determined at 1.96 and 1.65 Šresolution, respectively, are reported. The new ligand-free crystal belonged to space group C2, with unit-cell parameters a=73.5, b=54.3, c=62.7 Å, ß=118.8°. The new ligand-free structure revealed an open conformation that differed from the previously determined conformation, with an r.m.s.d on Cα atoms of 1.4 Å. The new crystal of the complex with the two-substrate-mimicking inhibitor P1,P5-bis(adenosine-5'-)pentaphosphate (Ap5A) belonged to space group P1, with unit-cell parameters a=53.9, b=62.3, c=63.0 Å, α=101.9, ß=112.6, γ=89.9°. Despite belonging to the same space group as the previously reported crystal, the new Ap5A-bound crystal contains four molecules in the asymmetric unit, compared with two in the previous crystal, and shows slightly different lattice contacts. These results demonstrate that SpAdK can crystallize promiscuously in different forms and that the open structure is flexible in conformation.

Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.

Streptococcus pneumoniae (pneumococcus) infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs) are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S . pneumoniae (SpAdK) serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A). Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.