L1 Loop Research Articles

Characterization of a promiscuous DNA sulfur binding domain and application in site-directed RNA base editing.

Phosphorothioate (PT)-modification was discovered in prokaryotes and is involved in many biological functions such as restriction-modification systems. PT-modification can be recognized by the sulfur binding domains (SBDs) of PT-dependent restriction endonucleases, through coordination with the sulfur atom, accompanied by interactions with the DNA backbone and bases. The unique characteristics of PT recognition endow SBDs with the potential to be developed into gene-targeting tools, but previously reported SBDs display sequence-specificity for PT-DNA, which limits their applications. In this work, we identified a novel sequence-promiscuous SBDHga from Hahella ganghwensis. We solved the crystal structure of SBDHga complexed with PT-DNA substrate to 1.8 Å resolution and revealed the recognition mechanism. A shorter L4 loop of SBDHga interacts with the DNA backbone, in contrast with previously reported SBDs, which interact with DNA bases. Furthermore, we explored the feasibility of using SBDHga and a PT-oligonucleotide as targeting tools for site-directed adenosine-to-inosine (A-to-I) RNA editing. A GFP non-sense mutant RNA was repaired at about 60% by harnessing a chimeric SBD-hADAR2DD (deaminase domain of human adenosine deaminase acting on RNA), comparable with currently available RNA editing techniques. This work provides insights into understanding the mechanism of sequence-specificity for SBDs and for developing new tools for gene therapy.

Elucidating the Mechanisms of R248Q Mutation-Enhanced p53 Aggregation and Its Inhibition by Resveratrol.

Aggregation of p53 mutants can result in loss-of-function, gain-of-function, and dominant-negative effects that contribute to tumor growth. Revealing the mechanisms underlying mutation-enhanced p53 aggregation and dissecting how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations are fundamentally important for the development of novel therapeutics for p53 aggregation-associated cancers. A recent experimental study shows that resveratrol (RSV) has an inhibitory effect on the aggregation of hot-spot R248Q mutant of the p53 core domain (p53C), while pterostilbene (PT) exhibits a relatively poor inhibitory efficacy. However, the conformational properties of the R248Q mutant leading to its enhanced aggregation propensity and the inhibitory mechanism of RSV against p53C aggregation are not well understood. Herein, we performed extensive all-atom molecular dynamics simulations on R248Q p53C in the absence and presence of RSV/PT, as well as wild-type (WT) p53C. Our simulations reveal that loop L3, where the mutation resides, remains compact in WT p53C, while it becomes extended in the R248Q mutant. The extension of loop L3 weakens the interactions between loop L3 and two crucial aggregation-prone regions (APRs) of p53C, leading to impaired interactions within the APRs and their structural destabilization as well as p53C. The destabilized APRs in the R248Q mutant are more exposed than in WT p53C, which is conducive to p53C aggregation. RSV has a higher preference to bind to R248Q p53C than PT. This binding not only stabilizes loop L3 of R248Q mutant to its WT-like conformation, preventing L3-extension-caused APRs' destabilization but also reduces APRs' solvent exposure, thereby inhibiting R248Q p53C aggregation. However, PT exhibits a lower hydrogen-bonding capability and a higher self-association propensity, which would lead to a reduced p53C binding and a weakened inhibitory effect on R248Q mutant aggregation. Our study provides mechanistic insights into the R248Q mutation-enhanced aggregation propensity and RSV's potent inhibition against R248Q p53C aggregation.

Structure and Function of Kdac1, a Class II Deacetylase from the Multidrug-Resistant Pathogen Acinetobacter baumannii.

Proteomics studies indicate that 10% of proteins in the opportunistic pathogen Acinetobacter baumannii are acetylated, suggesting that lysine acetyltransferases and deacetylases function to maintain and regulate a robust bacterial acetylome. As the first step in exploring these fascinating prokaryotic enzymes, we now report the preparation and characterization of the lysine deacetylase Kdac1. We show that Kdac1 catalyzes the deacetylation of free acetyllysine and acetyllysine tetrapeptide assay substrates, and we also report the X-ray crystal structures of unliganded Kdac1 as well as its complex with the hydroxamate inhibitor Citarinostat. Kdac1 is a tetramer in solution and in the crystal; the crystal structure reveals that the L1 loop functions to stabilize quaternary structure, forming inter-subunit hydrogen bonds and salt bridges around a central arginine residue (R30). Surprisingly, the L1 loop partially blocks entry to the active site, but it is sufficiently flexible to allow for the binding of two Citarinostat molecules in the active site. The L12 loop is also important for maintaining quaternary structure; here, a conserved arginine (R278) accepts hydrogen bonds from the backbone carbonyl groups of residues in an adjacent monomer. Structural comparisons with two other prokaryotic lysine deacetylases reveal conserved residues in the L1 and L12 loops that similarly support tetramer assembly. These studies provide a structural foundation for understanding enzymes that regulate protein function in bacteria through reversible lysine acetylation, serving as a first step in the exploration of these enzymes as possible targets for the development of new antibiotics.

Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

Alternating L4 loop architecture of the bacterial polysaccharide co-polymerase WzzE

Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the environment and are often polymerized by the inner membrane bound Wzy-dependent pathway. By employing cryo-electron microscopy we show that WzzE, the co-polymerase component of this pathway that is responsible for the length modulation of the enterobacterial common antigen, is octameric with alternating up-down conformations of its L4 loops. The alternating up-down nature of these essential loops, located at the top of the periplasmic bell, are modulated by clashing helical faces between adjacent protomers that flank the L4 loops around the octameric periplasmic bell. This alternating arrangement and a highly negatively charged binding face create a dynamic environment in which the polysaccharide chain is extended, and suggest a ratchet-type mechanism for polysaccharide elongation.

Computational and Experimental Analyses for Pathogenicity Prediction of ACVRL1 Missense Variants in Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease caused by the defects of ALK1/ACVRL1 receptor signaling. In this study, we evaluated 25 recently identified ACVRL1 missense variants using multiple computational pathogenicity classifiers and experimentally characterized their signal transduction capacity. Three extracellular residue variants showed no detectable cell surface expression and impairment of bone morphogenetic protein 9 (BMP9) responsiveness of SMAD-dependent transcription in luciferase assays. Four variants with amino acid replacement in the motifs essential for the intracellular kinase function lost SMAD-dependent signaling. Most of other variations in the kinase domain also caused marked downregulation of signaling; however, two variants behaved as the wild-type ACVRL1 did, while computational classifiers predicted their functional abnormalities. Three-dimensional structure prediction using the ColabFold program supported the significance of the L45 loop and NANDOR domain of ACVRL1 for its association with SMAD1 and BMPR2, respectively, and the variations in these motifs resulted in the reduction of SMAD signaling. On the other hand, two of the GS domain variants maintained high signal transduction capacity, which did not accord with their computational pathogenicity prediction. These results affirm the requirement of a combinatory approach using computational and experimental analyses to accurately predict the pathogenicity of ACVRL1 missense variants in the HHT patients.

Structural and functional insights of a cold-adaptive β-glucosidase with very high glucose tolerance from Microbacterium sp. CIAB417

A gene glu1 (WP_243232135.1) coding for β-glucosidase from the genome of Microbacterium sp. CIAB417 was characterized for its cold adaptive nature and tolerance to high levels of glucose and ethanol. The phylogenetic analysis suggested the close association of glu1 with a similar gene from a mesophilic bacterium Microbacterium indicum. The purified recombinant GLU1 displayed its optimal activity and stability at pH 5 and temperature 30ᴼC. Additionally, the presence of L3 loop in GLU1 suggested its cold adaptive nature. The glucose tolerant Gate keeper residues (Leu 174 & Trp 169) with a distance of ∼ 6.953 Å between them was also predicted in GLU1. The GLU1 enzyme showed ≥ 95% and ≥ 40% relative activity in the presence of 5 M glucose and 20% ethanol. The Vmax, Km, and Kcat values of GLU1 for cellobiose substrate were observed to be 45.22 U/mg, 3.5 mM, and 41.0157 s-1, respectively. The GLU1 was found to be highly efficient in hydrolysis of celloologosaccharides (C2-C5), lactose and safranal picrocrocin into glucose. Hence, cold adaptive GLU1 with very high glucose and ethanol tolerance could be very useful in bio-refinery, dairy, and flavor industries.

Recurrent Campylobacter jejuni Infections with In Vivo Selection of Resistance to Macrolides and Carbapenems: Molecular Characterization of Resistance Determinants.

We present two independent cases of recurrent multidrug-resistant Campylobacter jejuni infection in immunocompromised hosts and the clinical challenges encountered due to the development of high-level carbapenem resistance. The mechanisms associated with this unusual resistance for Campylobacters were characterized. Initial macrolide and carbapenem-susceptible strains acquired resistance to erythromycin (MIC > 256mg/L), ertapenem (MIC > 32mg/L), and meropenem (MIC > 32mg/L) during treatment. Carbapenem-resistant isolates developed an in-frame insertion resulting in an extra Asp residue in the major outer membrane protein PorA, within the extracellular loop L3 that connects β-strands 5 and 6 and forms a constriction zone involved in Ca2+ binding. The isolates presenting the highest MIC to ertapenem exhibited an extra nonsynonymous mutation (G167A|Gly56Asp) at PorA's extracellular loop L1. IMPORTANCE Carbapenem susceptibility patterns suggest drug impermeability, related to either insertion and/or single nucleotide polymorphism (SNP) within porA. Similar molecular events occurring in two independent cases support the association of these mechanisms with carbapenem resistance in Campylobacter spp.

Occurrence and phylogenetic analysis of fowl adenovirus E in broiler flocks from Gaza Strip Palestine.

Inclusion-body hepatitis (IBH) and hydropericardium syndrome (HPS) are highly infectious diseases caused by fowl adenoviruses (FAdVs). IBH and HPS cause major economic losses in poultry production. IBH is caused by multiple FAdV serotypes such as FAdV-11, FAdV8a, and FAdV8b; while HPS is mainly caused by the FAdV-4 serotype. In 2018, we detected FAdVs in West Bank - Palestine for the first time. This study aims to monitor the emergence of new FAdVs in broiler farms in Gaza Strip-Palestine in 2022. The clinical signs, necropsy, and histopathological findings associated with IBH in the suspected birds were recorded. Polymerase chain reaction (PCR) was performed using primers matching the virus-encoded L1 loop of the hexon gene. The sequences of the L1 loop were analyzed and a phylogenetic tree was constructed and compared with the related FAdV field isolates and reference strains from different regions of the world available in GenBank. The infected broiler displayed FAdVs-induced clinical symptoms and pathological lesions with mortality rates ranging from 20-46%. The L1 loop sequences from the infected flocks were submitted to GenBank with accession numbers ON638995, ON872150, and ON872151. The identified L1 loop gene has high nucleotide homology (96.7-97.9%) to the highly pathogenic FAdV E serotype 8b strain FAdV_isolate_04-53357-122_Canada_2007 (GenBank: EF685489) and 94.5-94.6% to FAdV_10_Belgium_2010 isolate 11-15941 (GenBank: AF339924.1). Furthermore, the phylogenetic analysis indicated that they belong to FAdV-E serotype 8b. Our study reports the emergence of FAdV-E causing IBH disease in broiler chickens for the first time in Gaza in Palestine.

The sphingolipids ceramide and inositol phosphorylceramide protect the Leishmania major membrane from sterol-specific toxins

The accessibility of sterols in mammalian cells to exogenous sterol-binding agents has been well-described previously, but sterol accessibility in distantly related protozoa is unclear. The human pathogen Leishmania major uses sterols and sphingolipids distinct from those used in mammals. Sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids, but the surface exposure of ergosterol in Leishmania remains unknown. Here, we used flow cytometry to test the ability of the L.major sphingolipids inositol phosphorylceramide (IPC) and ceramide to shelter ergosterol by preventing binding of the sterol-specific toxins streptolysin O and perfringolysin O and subsequent cytotoxicity. In contrast to mammalian systems, we found that Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, we show that IPC reduced cytotoxicity and that ceramide reduced perfringolysin O- but not streptolysin O-mediated cytotoxicity in cells. Furthermore, we demonstrate ceramide sensing was controlled by the toxin L3 loop, and that ceramide was sufficient to protect L.major promastigotes from the anti-leishmaniasis drug amphotericin B. Based on these results, we propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major could serve as a genetically tractable protozoan model organism for understanding toxin-membrane interactions.

Correlating histone acetylation with nucleosome core particle dynamics and function

Epigenetic modifications of chromatin play a critical role in regulating the fidelity of the genetic code and in controlling the translation of genetic information into the protein components of the cell. One key posttranslational modification is acetylation of histone lysine residues. Molecular dynamics simulations, and to a smaller extent experiment, have established that lysine acetylation increases the dynamics of histone tails. However, a systematic, atomic resolution experimental investigation of how this epigenetic mark, focusing on one histone at a time, influences the structural dynamics of the nucleosome beyond the tails, and how this translates into accessibility of protein factors such as ligases and nucleases, has yet to be performed. Herein, using NMR spectroscopy of nucleosome core particles (NCPs), we evaluate the effects of acetylation of each histone on tail and core dynamics. We show that for histones H2B, H3, and H4, the histone core particle dynamics are little changed, even though the tails have increased amplitude motions. In contrast, significant increases to H2A dynamics are observed upon acetylation of this histone, with the docking domain and L1 loop particularly affected, correlating with increased susceptibility of NCPs to nuclease digestion and more robust ligation of nicked DNA. Dynamic light scattering experiments establish that acetylation decreases inter-NCP interactions in a histone-dependent manner and facilitates the development of a thermodynamic model for NCP stacking. Our data show that different acetylation patterns result in nuanced changes to NCP dynamics, modulating interactions with other protein factors, and ultimately controlling biological output.

Genetic diversity and bioinformatic analysis in the L1 gene of HPV genotypes 31, 33, and 58 circulating in women with normal cervical cytology

BackgroundHPV-31, -33, and -58, along with HPV-45 and -52, account for almost 11% of HPV-associated cancers. Our previous studies showed that after HPV-16 and -51, HPV-58 was common and HPV-31 was as frequent as HPV-18 among Iranian women with normal cytology. Hence, in this study, we aimed to investigate the intra-type variations in L1 genes of HPV-58, -31, and -33 to find the predominant lineages circulating in women with normal cytology.MethodsComplete coding sequencing of the L1 gene was amplified and nucleotide and amino acid sequences were compared to those of the references. The selective pressure on L1 protein and whether the variations of the L1 genes embed in L1 loops, or N-glycosylated sites were also investigated.ResultsB1, A, and A1 (sub)lineages were common in the HPV-58, -33, and -31 samples, respectively. Ninety nucleotide mutations were observed. Twenty nine nucleotide changes corresponded to nonsynonymous substitutions in which seventeen mutations were located in L1 loops. Only one codon position in HPV-58 sequences was found as the positive selection. No difference was observed in N-glycosylation sites between reference and understudied amino acid sequences.ConclusionIn the current study, we reported, for the first time, the (sub) lineages, amino acid, and genetic diversity in the L1 gene of circulating HPV-58, -33, and -31, in women with normal cytology, in Iran. Such studies can not only have epidemiological values, but also aid to set vaccination programs.

Template Entrance Channel as Possible Allosteric Inhibition and Resistance Site for Quinolines Tricyclic Derivatives in RNA Dependent RNA Polymerase of Bovine Viral Diarrhea Virus

The development of potent non-nucleoside inhibitors (NNIs) could be an alternate strategy to combating infectious bovine viral diarrhea virus (BVDV), other than the traditional vaccination. RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral replication; therefore, it is one of the primary targets for countermeasures against infectious diseases. The reported NNIs, belonging to the classes of quinolines (2h: imidazo[4,5-g]quinolines and 5m: pyrido[2,3-g] quinoxalines), displayed activity in cell-based and enzyme-based assays. Nevertheless, the RdRp binding site and microscopic mechanistic action are still elusive, and can be explored at a molecular level. Here, we employed a varied computational arsenal, including conventional and accelerated methods, to identify quinoline compounds' most likely binding sites. Our study revealed A392 and I261 as the mutations that can render RdRp resistant against quinoline compounds. In particular, for ligand 2h, mutation of A392E is the most probable mutation. The loop L1 and linker of the fingertip is recognized as a pivotal structural determinant for the stability and escape of quinoline compounds. Overall, this work demonstrates that the quinoline inhibitors bind at the template entrance channel, which is governed by conformational dynamics of interactions with loops and linker residues, and reveals structural and mechanistic insights into inhibition phenomena, for the discovery of improved antivirals.

Non-active Site Residue in Loop L4 Alters Substrate Capture and Product Release in d-Arginine Dehydrogenase.

Numerous studies demonstrate that enzymes undergo multiple conformational changes during catalysis. The malleability of enzymes forms the basis for allosteric regulation: residues located far from the active site can exert long-range dynamical effects on the active site residues to modulate catalysis. The structure of Pseudomonas aeruginosa d-arginine dehydrogenase (PaDADH) shows four loops (L1, L2, L3, and L4) that span the substrate and the FAD-binding domains. Loop L4 comprises residues 329-336, spanning over the flavin cofactor. The I335 residue on loop L4 is ∼10 Å away from the active site and ∼3.8 Å from N(1)-C(2)═O atoms of the flavin. In this study, we used molecular dynamics and biochemical techniques to investigate the effect of the mutation of I335 to histidine on the catalytic function of PaDADH. Molecular dynamics showed that the conformational dynamics of PaDADH are shifted to a more closed conformation in the I335H variant. In agreement with an enzyme that samples more in a closed conformation, the kinetic data of the I335H variant showed a 40-fold decrease in the rate constant of substrate association (k1), a 340-fold reduction in the rate constant of substrate dissociation from the enzyme-substrate complex (k2), and a 24-fold decrease in the rate constant of product release (k5), compared to that of the wild-type. Surprisingly, the kinetic data are consistent with the mutation having a negligible effect on the reactivity of the flavin. Altogether, the data indicate that the residue at position 335 has a long-range dynamical effect on the catalytic function in PaDADH.

The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region.

Mammalian myxovirus resistance (Mx) proteins are interferon-induced, large dynamin-like GTPases with a broad antiviral spectrum. Here, we analyzed the antiviral activity of selected mammalian Mx1 proteins against Thogoto virus (THOV). Of those, equine Mx1 (eqMx1) showed antiviral activity comparable to that of the human MX1 gene product, designated huMxA, whereas most Mx1 proteins were antivirally inactive. We previously demonstrated that the flexible loop L4 protruding from the stalk domain of huMxA, and especially the phenylalanine at position 561 (F561), determines its antiviral specificity against THOV (P. S. Mitchell, C. Patzina, M. Emerman, O. Haller, et al., Cell Host Microbe 12:598-604, 2012, https://doi.org/10.1016/j.chom.2012.09.005). However, despite the similar antiviral activity against THOV, the loop L4 sequence of eqMx1 substantially differs from the one of huMxA. Mutational analysis of eqMx1 L4 identified a tryptophan (W562) and the adjacent glycine (G563) as critical antiviral determinants against THOV, whereas the neighboring residues could be exchanged for nonpolar alanines without affecting the antiviral activity. Further mutational analyses revealed that a single bulky residue at position 562 and the adjacent tiny residue G563 were sufficient for antiviral activity. Moreover, this minimal set of L4 amino acids transferred anti-THOV activity to the otherwise inactive bovine Mx1 (boMx1) protein. Taken together, our data suggest a fairly simple architecture of the antiviral loop L4 that could serve as a mutational hot spot in an evolutionary arms race between Mx-escaping viral variants and their hosts. IMPORTANCE Most mammals encode two paralogs of the interferon-induced Mx proteins: Mx1, with antiviral activity largely against RNA viruses, like orthomyxoviruses and bunyaviruses; and Mx2, which is antivirally active against HIV-1 and herpesviruses. The human Mx1 protein, also called huMxA, is the best-characterized example of mammalian Mx1 proteins and was recently shown to prevent zoonotic virus transmissions. To evaluate the antiviral activity of other mammalian Mx1 proteins, we used Thogoto virus, a tick-transmitted orthomyxovirus, which is efficiently blocked by huMxA. Interestingly, we detected antiviral activity only with equine Mx1 (eqMx1) but not with other nonprimate Mx1 proteins. Detailed functional analysis of eqMx1 identified amino acid residues in the unstructured loop L4 of the stalk domain critical for antiviral activity. The structural insights of the present study explain the unique position of eqMx1 antiviral activity within the collection of nonhuman mammalian Mx1 proteins.

Exploring the structural and dynamic differences between human carnosinase I (CN1) and II (CN2).

Human carnosinases (CNs) are dimeric dipeptidases in the metallopeptidase M20 family. Two isoforms of carnosinases (Zn2+ -containing carnosinase 1 (CN1) found in serum and Mn2+ -carnosinase 2 (CN2) in tissue) were identified. Both CNs cleave histidine-containing (Xaa-His) dipeptides such as carnosine where CN2 was found to accept a broader spectrum of substrates. A loss of CN function, resulting in a high carnosine concentration, reduces risk for diabetes and neurological disorders. Although several studies on CN activities and its Michaelis complex were conducted, all shed the light on CN1 activity where the CN2 data is limited. Also, the molecular details on CN1 and CN2 similarity and dissimilarity in structure and function remain unclear. Thus, in this work, molecular dynamics (MD) simulations were employed to study structure and dynamics of human CN1 and CN2 in comparison. The results show that the different catalytic ability of both CNs is due to their pocket size and environment. CN2 can accept a wider range of substrate due to the wider mouth of a binding pocket. The L1 loop seems to play a role in gating activity. Comparing to CN2, CN1 provides more electronegative entrance, more wettability, and higher stability of catalytic metal ion-pair in the active site which allow more efficient water-mediated catalysis. The microscopic understanding obtained here can serve as a basis for CN inhibition strategies resulting in higher carnosine levels and consequently mitigating complications associated with diseases such as diabetes and neurological disorder.

Xanthohumol Interferes with the Activation of TGF-β Signaling in the Process Leading to Intestinal Fibrosis

Fibrosis has various biological processes and affects almost every organ, especially in patients with inflammatory bowel disease, including Crohn's disease, who experience discomfort caused by intestinal fibrosis, which is a problem that needs to be resolved. TGF-β signaling is known to act as a key regulator of intestinal fibrosis, and its modulation could be an excellent candidate for fibrosis therapy. Xanthohumol (XN) has various effects, including anti-inflammation and anti-cancer; however, the detailed mechanism of TGF-β signaling has not yet been studied. The purpose of this study was to investigate the mechanism underlying the anti-fibrotic effect of XN on TGF-β1-induced intestinal fibrosis using primary human intestinal fibroblasts (HIFs). In this study, to check the anti-fibrotic effects of XN on intestinal fibrosis, we assessed the expression of fibrosis-related genes in TGF-β1-stimulated HIFs by qPCR, immunoblotting, and immunofluorescence staining. As a result, XN showed the ability to reduce the expression of fibrosis-associated genes increased by TGF-β1 treatment in HIFs and restored the cell shape altered by TGF-β1. In particular, XN repressed both NF-κB- and Smad-binding regions in the α-SMA promoter, which is important in fibrosis. In addition, XN inhibited NF-κB signaling, including phosphorylated-IkBα and cyclooxygenase-2 expression, and TNF-α-stimulated transcriptional activity of NF-κB. XN attenuated TGF-β1-induced phosphorylation of Smad2 and Smad3, and the transcriptional activity of CAGA. Particularly, XN interfered with the binding of TGF-Receptor I (TβRI) and Smad3 by binding to the kinase domain of the L45 loop of TβRI, thereby confirming that the fibrosis mechanism did not proceed further. In conclusion, XN has an inhibitory effect on TGF-β1-induced intestinal fibrosis in HIFs, significantly affecting TGF-β/Smad signaling.

Conformational Dynamics of Loop L3 in OmpF: Implications toward Antibiotic Translocation and Voltage Gating.

In the present work, we delineate the molecular mechanism of a bulky antibiotic permeating through a bacterial channel and uncover the role of conformational dynamics of the constriction loop in this process. Using the temperature accelerated sliced sampling approach, we shed light onto the dynamics of the L3 loop, in particular the F118 to S125 segment, at the constriction regions of the OmpF porin. We complement the findings with single channel electrophysiology experiments and applied-field simulations, and we demonstrate the role of hydrogen-bond stabilization in the conformational dynamics of the L3 loop. A molecular mechanism of permeation is put forward wherein charged antibiotics perturb the network of stabilizing hydrogen-bond interactions and induce conformational changes in the L3 segment, thereby aiding the accommodation and permeation of bulky antibiotic molecules across the constriction region. We complement the findings with single channel electrophysiology experiments and demonstrate the importance of the hydrogen-bond stabilization in the conformational dynamics of the L3 loop. The generality of the present observations and experimental results regarding the L3 dynamics enables us to identify this L3 segment as the source of gating. We propose a mechanism of OmpF gating that is in agreement with previous experimental data that showed the noninfluence of cysteine double mutants that tethered the L3 tip to the barrel wall on the OmpF gating behavior. The presence of similar loop stabilization networks in porins of other clinically relevant pathogens suggests that the conformational dynamics of the constriction loop is possibly of general importance in the context of antibiotic permeation through porins.

An evolutionarily conserved N-terminal leucine is essential for MX1 GTPase antiviral activity against different families of RNA viruses

Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins.

Development of Fluorinated Peptoid-Based Histone Deacetylase (HDAC) Inhibitors for Therapy-Resistant Acute Leukemia.

Using a microwave-assisted protocol, we synthesized 16 peptoid-capped HDAC inhibitors (HDACi) with fluorinated linkers and identified two hit compounds. In biochemical and cellular assays, 10h stood out as a potent unselective HDACi with remarkable cytotoxic potential against different therapy-resistant leukemia cell lines. 10h demonstrated prominent antileukemic activity with low cytotoxic activity toward healthy cells. Moreover, 10h exhibited synergistic interactions with the DNA methyltransferase inhibitor decitabine in AML cell lines. The comparison of crystal structures of HDAC6 complexes with 10h and its nonfluorinated counterpart revealed a similar occupation of the L1 loop pocket but slight differences in zinc coordination. The substitution pattern of the acyl residue turned out to be crucial in terms of isoform selectivity. The introduction of an isopropyl group onto the phenyl ring provided the highly HDAC6-selective inhibitor 10p, which demonstrated moderate synergy with decitabine and exceeded the HDAC6 selectivity of tubastatin A.