| 1. |
- Kauppi, Anna, 1971-, et al.
(författare)
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Targeting bacterial Virulence : Inhibitors of type III secretion in Yersinia
- 2003
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Ingår i: Chemistry and Biology. - 1074-5521 .- 1879-1301. ; 10:3, s. 241-249
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Tidskriftsartikel (refereegranskat)abstract
- Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.
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| 2. |
- Tjernberg, L O, et al.
(författare)
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Amyloid beta-peptide polymerization studied using fluorescence correlation spectroscopy
- 1999
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Ingår i: Chemistry and Biology. - 1074-5521 .- 1879-1301. ; 6:1, s. 53-62
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Tidskriftsartikel (refereegranskat)abstract
- Background: The accumulation of fibrillar deposits of amyloid beta-peptide (A beta) in brain parenchyma and cerebromeningeal blood vessels is a key step in the pathogenesis of Alzheimer's disease. In this report, polymerization of A beta was studied using fluorescence correlation spectroscopy (FCS), a technique capable of detecting small molecules and large aggregates simultaneously in solution. Results: The polymerization of A beta dissolved in Tris-buffered saline, pH 7.4, occurred above a critical concentration of 50 mu M and proceeded from monomers/dimers into two discrete populations of large aggregates, without any detectable amount of oligomers. The aggregation showed very high cooperativity and reached a maximum after 40 min, followed by an increase in the amount of monomers/dimers and a decrease in the size of the large aggregates. Electron micrographs of samples prepared at the time for maximum aggregation showed a mixture of an amorphous network and short diffuse fibrils, whereas only mature amyloid fibrils were detected after one day of incubation. The aggregation was reduced when A beta was incubated in the presence of A beta ligands, oligopeptides previously shown to inhibit fibril formation, and aggregates were partly dissociated after the addition of the ligands. Conclusions: The polymerization of A beta is a highly cooperative process in which the formation of very large aggregates precedes the formation of fibrils. The entire process can be inhibited and, at least in early stages, partly reversed by A beta ligands.
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| 3. |
- Andersson, Emma K., et al.
(författare)
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Modulation of Curli Assembly and Pellicle Biofilm Formation by Chemical and Protein Chaperones
- 2013
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Ingår i: Chemistry and Biology. - : Elsevier. - 1074-5521 .- 1879-1301. ; 20:10, s. 1245-1254
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Tidskriftsartikel (refereegranskat)abstract
- Enteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by "chemical chaperones."
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| 4. |
- Andersson, Emma K., et al.
(författare)
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Small Molecule Disruption of B-subtilis Biofilms by Targeting the Amyloid Matrix
- 2013
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Ingår i: Chemistry and Biology. - : Cell Press. - 1074-5521 .- 1879-1301. ; 20:1, s. 5-7
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Small molecule inhibitors of amyloid aggregation have potential as treatment for a variety of conditions. In this issue of Chemistry & Biology, Romero and colleagues use amyloid-dependent B. subtilis biofilm formation to screen for amyloid inhibitors, identifying compounds that not only inhibit B. subtilis biofilm formation but also ones that disrupt preformed biofilms.
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| 5. |
- Andersson, Theresa, et al.
(författare)
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The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors
- 2005
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 12:11, s. 1245-1252
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Tidskriftsartikel (refereegranskat)abstract
- Several receptors for human carbonic anhydrase II (HCAII) have been prepared by covalently attaching benzenesulfonamide carboxylates via aliphatic aminocarboxylic acid spacers of variable length to the side chain of a lysine residue in a designed 42 residue helix-loop-helix motif. The sulfonamide group binds to the active site zinc ion of human carbonic anhydrase II located in a 15 Å deep cleft. The dissociation constants of the receptor-HCAII complexes were found to be in the range from low micromolar to better than 20 nM, with the lowest affinities found for spacers with less than five methylene groups and the highest affinity found for the spacer with seven methylene groups. The results suggest that the binding is a cooperative event in which both the sulfonamide residue and the helix-loop-helix motif contribute to the overall affinity.
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| 6. |
- Anscombe, Elizabeth, et al.
(författare)
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Identification and Characterization of an Irreversible Inhibitor of CDK2
- 2015
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 22:9, s. 1159-1164
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Tidskriftsartikel (refereegranskat)abstract
- Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclo-hexylmethoxy)-9H-purin-2-yl) amino) benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl) phenyl)-9H- purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds.
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| 7. |
- Braesel, Jana, et al.
(författare)
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Three Redundant Synthetases Secure Redox-Active Pigment Production in the Basidiomycete Paxillus involutus.
- 2015
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 22:10, s. 1325-1334
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Tidskriftsartikel (refereegranskat)abstract
- The symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton chemistry. Their precise biosynthetic pathways, however, remain obscure. Using a combination of biochemical, genetic, and transcriptomic analyses, in addition to stable-isotope labeling with synthetic precursors, we show that atromentin is the key intermediate. Atromentin is made by tridomain synthetases of high similarity: InvA1, InvA2, and InvA5. An inactive atromentin synthetase, InvA3, gained activity after a domain swap that replaced its native thioesterase domain with that of InvA5. The found degree of multiplex biosynthetic capacity is unprecedented with fungi, and highlights the great importance of the metabolite for the producer.
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| 8. |
- Doak, Bradley Croy, et al.
(författare)
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Oral Druggable Space beyond the Rule of 5 : Insights from Drugs and Clinical Candidates
- 2014
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 21:9, s. 1115-1142
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Forskningsöversikt (refereegranskat)abstract
- The rule of 5 (Ro5) is a set of in silico guidelines applied to drug discovery to prioritize compounds with an increased likelihood of high oral absorption. It has been influential in reducing attrition due to poor pharmacokinetics over the last 15 years. However, strict reliance on the Ro5 may have resulted in lost opportunities, particularly for difficult targets. To identify opportunities for oral drug discovery beyond the Ro5 (bRo5), we have comprehensively analyzed drugs and clinical candidates with molecular weight (MW) > 500 Da. We conclude that oral drugs are found far bRo5 and properties such as intramolecular hydrogen bonding, macrocyclization, dosage, and formulations can be used to improve bRo5 bioavailability. Natural products and structure-based design, often from peptidic leads, are key sources for oral bRo5 drugs. These insights should help guide the design of oral drugs in bRo5 space, which is of particular interest for difficult targets.
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| 9. |
- Haeggstrom, JZ, et al.
(författare)
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Resolving resolvins
- 2013
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Ingår i: Chemistry & biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 20:2, s. 138-140
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Tidskriftsartikel (refereegranskat)
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| 10. |
- Hansson, Magnus, et al.
(författare)
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Bioengineering and Semisynthesis of an Optimized Cyclophilin Inhibitor for Treatment of Chronic Viral Infection.
- 2015
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 22:2, s. 285-292
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Tidskriftsartikel (refereegranskat)abstract
- Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products, which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed nonribosomal peptide/polyketide origin, to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of hepatitis B virus, hepatitis C virus (HCV), and HIV-1 replication, shows minimal inhibition of major drug transporters, and has a high barrier to generation of both HCV and HIV-1 resistance.
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| 11. |
- Hegazy, Usama M., et al.
(författare)
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Replacement surgery with unnatural amino acids in the lock-and-key joint of glutathione transferase subunits
- 2006
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 13:9, s. 929-936
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Tidskriftsartikel (refereegranskat)abstract
- Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.
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| 12. |
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| 13. |
- Iwamoto, Mari, et al.
(författare)
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A General Chemical Method to Regulate Protein Stability in the Mammalian Central Nervous System
- 2010
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 17:9, s. 981-988
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Tidskriftsartikel (refereegranskat)abstract
- The ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. We have developed a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of the Escherichia coil dihydrofolate reductase (ecDHFR) were engineered to be degraded, and, when this destabilizing domain is fused to a protein of interest, its instability is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. A small-molecule ligand trimethoprim (TMP) stabilizes the destabilizing domain in a rapid, reversible, and dose-dependent manner, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood-brain barrier enables the tunable regulation of proteins expressed in the mammalian central nervous system.
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| 14. |
- Jones, Sarah, et al.
(författare)
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Characterisation of bioactive cell penetrating peptides from human Cytochrome c : protein mimicry and the development of a novel apoptogenic agent
- 2010
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 17:7, s. 735-744
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Tidskriftsartikel (refereegranskat)abstract
- Cell penetrating peptides (CPPs) with intrinsic biological activities offer a novel strategy for the modulation of intracellular events. QSAR analysis identified CPPs within human cytochrome c. Two such sequences, Cyt c77–101 and Cyt c86–101, induced tumor cell apoptosis, thus mimicking the role of Cyt c as a key regulator of programmed cell death. Quantitative analyses confirmed that Cyt c77–101 is an extremely efficient CPP. Thus, Cyt c77–101 was selected for modification to incorporate target-specific peptidyl motifs. Chimeric N-terminal extension with a target mimetic of FG nucleoporins significantly enhanced the apoptogenic potency of Cyt c77–101 to a concentration readily achievable in vivo. Moreover, this construct, Nup153-Cyt c, facilitates the dramatic redistribution of nuclear pore complex proteins and thus propounds the nuclear pore complex as a novel target for the therapeutic induction of apoptosis.
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| 15. |
- Kalén, Mattias, et al.
(författare)
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Combination of reverse and chemical genetic screens reveals angiogenesis inhibitors and targets.
- 2009
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Ingår i: Chemistry & biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 16:4, s. 432-41
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Tidskriftsartikel (refereegranskat)abstract
- We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.
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| 16. |
- Mate, Diana, et al.
(författare)
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Blood tolerant laccase by directed evolution
- 2013
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Ingår i: Chemistry and Biology. - : Elsevier. - 1074-5521 .- 1879-1301. ; 20:2, s. 223-231
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Tidskriftsartikel (refereegranskat)abstract
- High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnol. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific compn. of human blood (above neutral pH, high chloride concn.) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chem. syntheses, and detoxification.
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| 17. |
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| 18. |
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| 19. |
- Park, S., et al.
(författare)
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Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations
- 2005
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 12:1, s. 45-54
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Tidskriftsartikel (refereegranskat)abstract
- Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach-random mutagenesis within the substrate binding site-is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.
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| 20. |
- Patton, Gregory C., et al.
(författare)
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Cofactor mobility determines reaction outcome in the IMPDH and GMPR (beta-alpha)(8) barrel enzymes
- 2011
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Ingår i: Nature Chemical Biology. - 1552-4450 .- 1552-4469. ; 7, s. 950-958
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Tidskriftsartikel (refereegranskat)abstract
- Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP(star) as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 angstrom from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.
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| 21. |
- Rosengren, K. Johan, et al.
(författare)
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How bugs make lassos
- 2009
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 16:12, s. 1211-1212
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Tidskriftsartikel (refereegranskat)
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| 22. |
- Shaw, Jeffrey J., et al.
(författare)
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A Role for the 2' OH of peptidyl-tRNA substrate in peptide release on the ribosome revealed through RF-mediated rescue
- 2012
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Ingår i: Chemistry and Biology. - 1074-5521 .- 1879-1301. ; 19:8, s. 983-993
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Tidskriftsartikel (refereegranskat)abstract
- The 2' OH of the peptidyl-tRNA substrate is thought to be important for catalysis of both peptide bond formation and peptide release in the ribosomal active site. The release reaction also specifically depends on a release factor protein (RF) to hydrolyze the ester linkage of the peptidyl-tRNA upon recognition of stop codons in the A site. Here, we demonstrate that certain amino acid substitutions (in particular those containing hydroxyl or thiol groups) in the conserved GGQ glutamine of release factor RF1 can rescue defects in the release reaction associated with peptidyl-tRNA substrates lacking a 2' OH. We explored this rescue effect through biochemical and computational approaches that support a model where the 2' OH of the P-site substrate is critical for orienting the nucleophile in a hydrogen-bonding network productive for catalysis.
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| 23. |
- Shaw, Jeffrey J., et al.
(författare)
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A Role for the 2 ' OH of Peptidyl-tRNA Substrate in Peptide Release on the Ribosome Revealed through RF-Mediated Rescue
- 2012
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 19:8, s. 983-993
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Tidskriftsartikel (refereegranskat)abstract
- The 2' OH of the peptidyl-tRNA substrate is thought to be important for catalysis of both peptide bond formation and peptide release in the ribosomal active site. The release reaction also specifically depends on a release factor protein (RF) to hydrolyze the ester linkage of the peptidyl-tRNA upon recognition of stop codons in the A site. Here, we demonstrate that certain amino acid substitutions (in particular those containing hydroxyl or thiol groups) in the conserved GGQ glutamine of release factor RF1 can rescue defects in the release reaction associated with peptidyl-tRNA substrates lacking a 2' OH. We explored this rescue effect through biochemical and computational approaches that support a model where the 2' OH of the P-site substrate is critical for orienting the nucleophile in a hydrogen-bonding network productive for catalysis.
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| 24. |
- Tanaka, Gen, et al.
(författare)
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CXCR4 Stimulates Macropinocytosis : Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV
- 2012
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 19:11, s. 1437-1446
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Tidskriftsartikel (refereegranskat)abstract
- CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.
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| 25. |
- Tatarek-Nossol, Marianna, et al.
(författare)
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Inhibition of hIAPP amyloid-fibril formation and apoptotic cell death by a designed hIAPP amyloid-core-containing hexapeptide
- 2005
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 12:7, s. 797-809
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Tidskriftsartikel (refereegranskat)abstract
- The pathogenesis of type II diabetes is associated with the aggregation of the 37-residue human islet amyloid polypeptide (hIAPP) into cytotoxic β sheet aggregates and fibrils. We have recently shown that introduction of two N-methyl rests in the β sheet- and amyloid-core-containing sequence hIAPP(22-27), or NFGAIL converted this amyloidogenic and cytotoxic sequence into nonamyloidogenic and noncytotoxic NF(N-Me)GA(N-Me)IL. Here, we show that NF(N-Me)GA(N-Me)IL is able to bind with high-affinity full-length hIAPP and to inhibit its fibrillogenesis. NF(N-Me)GA(N-Me)IL also inhibits hIAPP-mediated apoptotic β cell death. By contrast, unmodified NFGAIL does not inhibit hIAPP amyloidogenesis and cytotoxicity, suggesting that N-methylation conferred on NFGAIL the properties of NF(N-Me)GA(N-Me)IL. These results support the concept that rational N-methylation of hIAPP amyloid-core sequences may be a valuable strategy to design pancreatic-amyloid diagnostics and therapeutics for type II diabetes. ©2005 Elsevier Ltd All rights reserved.
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| 26. |
- Tholander, Fredrik, et al.
(författare)
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Structure-Based Dissection of the Active Site Chemistry of Leukotriene A4 Hydrolase: Implications for M1 Aminopeptidases and Inhibitor Design
- 2008
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 15:9, s. 920-929
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Tidskriftsartikel (refereegranskat)abstract
- M1 aminopeptidases comprise a large family of biologically important zinc enzymes. We show that peptide turnover by the M1 prototype, leukotriene A4 hydrolase/aminopeptidase, involves a shift in substrate position associated with exchange of zinc coordinating groups, while maintaining the overall coordination geometry. The transition state is stabilized by residues conserved among M1 members and in the final reaction step, Glu-296 of the canonical zinc binding HEXXH motif shuffles a proton from the hydrolytic water to the leaving group. Tripeptide substrates bind along the conserved GXMEN motif, precisely occupying the distance between Glu-271 and Arg-563, whereas the Arg specificity is governed by a narrow S1 pocket capped with Asp-375. Our data provide detailed insights to the active site chemistry of M1 aminopeptidases and will aid in the development of novel enzyme inhibitors.
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| 27. |
- Verdurmen, Wouter P. R., et al.
(författare)
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Preferential Uptake of L- versus D-Amino Acid Cell-Penetrating Peptides in a Cell Type-Dependent Manner
- 2011
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 18:8, s. 1000-1010
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Tidskriftsartikel (refereegranskat)abstract
- The use of protease-resistant D-peptides is a prominent strategy for overcoming proteolytic sensitivity in the use of cell-penetrating peptides (CPPs) as delivery vectors. So far, no major differences have been reported for the uptake of L- and D-peptides. Here we report that cationic L-CPPs are taken up more efficiently than their D-counterparts in MC57 fibrosarcoma and He La cells but not in Jurkat T leukemia cells. Reduced uptake of D-peptides co-occurred with persistent binding to heparan sulfates (HS) at the plasma membrane. In vitro binding studies of Land D-peptides with HS indicated similar binding affinities. Our results identify two key events in the uptake of CPPs: binding to HS chains and the initiation of internalization. Only the second event depends on the chirality of the CPP. This knowledge may be exploited for a stereochemistry-dependent preferential targeting of cells.
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| 28. |
- Vincents, Bjarne, et al.
(författare)
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The human protease inhibitor cystatin C is an activating cofactor for the streptococcal cysteine protease IdeS
- 2008
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 15:9, s. 960-968
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C is considered the physiologically most important inhibitor of endogenous papain-like cysteine proteases. We present here an unexpected function of cystatin C. Instead of acting as an inhibitor, cystatin C acts as a facultative, endogenous cofactor for the papain-like IgG-cleaving enzyme IdeS of the human pathogen Streptococcus pyogenes. IdeS activity is not dependent on cystatin C, but is significantly enhanced in the presence of cystatin C. We report a protease inhibitor that accelerates the activity of its putative target protease and a unique example of how a host protease inhibitor is "hijacked" by a bacterial protease to increase its activity. This finding has important implications for the view on protease-inhibitor interactions, and is relevant to consider in the therapeutic use of protease inhibitors.
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| 29. |
- Zhang, Wei, et al.
(författare)
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Structure-based redesign of GST A2-2 for enhanced catalytic efficiency with azathioprine
- 2012
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 19:3, s. 414-421
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.
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| 30. |
- Zumarraga, Miren, et al.
(författare)
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In vitro evolution of a fungal laccase in high concentrations of organic cosolvents
- 2007
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1879-1301 .- 1074-5521. ; 14:9, s. 1052-1064
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Tidskriftsartikel (refereegranskat)abstract
- Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase-R2 variant-was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.
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- Esbjörner Winters, Elin, 1978, et al.
(författare)
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Direct Observations of Amyloid beta Self-Assembly in Live Cells Provide Insights into Differences in the Kinetics of A beta(1-40) and A beta(1-42) Aggregation
- 2014
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521. ; 21:6, s. 732-742
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Tidskriftsartikel (refereegranskat)abstract
- Insight into how amyloid beta (A beta) aggregation occurs in vivo is vital for understanding the molecular pathways that underlie Alzheimer's disease and requires new techniques that provide detailed kinetic and mechanistic information. Using noninvasive fluorescence lifetime recordings, we imaged the formation of A beta(1-40) and A beta(1-42) aggregates in live cells. For both peptides, the cellular uptake via endocytosis is rapid and spontaneous. They are then retained in lysosomes, where their accumulation leads to aggregation. The kinetics of A beta(1-42) aggregation are considerably faster than those of A beta(1-40) and, unlike those of the latter peptide, show no detectable lag phase. We used superresolution fluorescence imaging to examine the resulting aggregates and could observe compact amyloid structures, likely because of spatial confinement within cellular compartments. Taken together, these findings provide clues as to how A beta aggregation may occur within neurons.
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- Bouabdallaoui, N, et al.
(författare)
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Growth differentiation factor-15 is not modified by sacubitril/valsartan and is an independent marker of risk in patients with heart failure and reduced ejection fraction: the PARADIGM-HF trial
- 2018
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Ingår i: European Journal of Heart Failure. - : Wiley. - 1388-9842 .- 1879-0844. ; 20:12, s. 1701-1709
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: Growth differentiation factor-15 (GDF-15) is associated with adverse prognosis in cardiovascular (CV) and non-CV diseases. We evaluated the association of GDF-15 with CV and non-CV outcomes in the PARADIGM-HF trial. METHODS AND RESULTS: In 1935 patients with heart failure and reduced ejection fraction (HFrEF) in PARADIGM-HF, median GDF-15 values were elevated and similar in sacubitril/valsartan and enalapril patients (1626 ng/L and 1690 ng/L, respectively). Diabetes, age, creatinine, high-sensitive troponin T, N-terminal pro-B-type natriuretic peptide, and New York Heart Association class III/IV were most strongly associated with elevated GDF-15 values (all P < 0.001) (adjusted R(2) = 0.3857). Baseline GDF-15 and changes in GDF-15 at both 1 month and 8 months (log-transformed) were associated with subsequent mortality and CV events. Each 20% increment in baseline GDF-15 value was associated with a higher risk of mortality [adjusted hazard ratio (HR) 1.13, 95% confidence interval (CI) 1.08-1.18, P < 0.001], the combined endpoint of CV death or hospitalization for heart failure (adjusted HR 1.09, 95% CI 1.05-1.14, P < 0.001) and heart failure death (adjusted HR 1.16, 95% CI 1.05-1.28, P < 0.001). Changes in GDF-15 were not influenced by assigned therapy (all P-values >/= 0.1). CONCLUSION: In patients with ambulatory HFrEF, GDF-15 is not modified by sacubitril/valsartan and is strongly associated with mortality and CV outcomes, suggesting that GDF-15 is a marker of poor outcomes in these patients. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01035255.
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| 39. |
- Meijers, Wouter C., et al.
(författare)
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Biomarkers and low risk in heart failure. Data from COACH and TRIUMPH
- 2015
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Ingår i: European Journal of Heart Failure. - : WILEY-BLACKWELL. - 1388-9842 .- 1879-0844. ; 17:12, s. 1271-1282
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Tidskriftsartikel (refereegranskat)abstract
- AimTraditionally, risk stratification in heart failure (HF) emphasizes assessment of high risk. We aimed to determine if biomarkers could identify patients with HF at low risk for death or HF rehospitalization. Methods and resultsThis analysis was a substudy of The Coordinating Study Evaluating Outcomes of Advising and Counselling in Heart Failure (COACH) trial. Enrolment of HF patients occurred before discharge. We defined low risk as the absence of death and/or HF rehospitalizations at 180days. We tested a diverse group of 29 biomarkers on top of a clinical risk model, with and without N-terminal pro-B-type natriuretic peptide (NT-proBNP), and defined the low risk biomarker cut-off at the 10th percentile associated with high positive predictive value. The best performing biomarkers together with NT-proBNP and cardiac troponin I (cTnI) were re-evaluated in a validation cohort of 285 HF patients. Of 592 eligible COACH patients, the mean (SD) age was 71 (+/- 11) years and median (IQR) NT-proBNP was 2521 (1301-5634) pg/mL. Logistic regression analysis showed that only galectin-3, fully adjusted, was significantly associated with the absence of events at 180days (OR 8.1, 95% confidence interval 1.06-50.0, P=0.039). Galectin-3, showed incremental value when added to the clinical risk model without NT-proBNP (increase in area under the curve from 0.712 to 0.745, P=0.04). However, no biomarker showed significant improvement by net reclassification improvement on top of the clinical risk model, with or without NT-proBNP. We confirmed our results regarding galectin-3, NT-proBNP, and cTnI in the independent validation cohort. Conclusion We describe the value of various biomarkers to define low risk, and demonstrate that galectin-3 identifies HF patients at (very) low risk for 30-day and 180-day mortality and HF rehospitalizations after an episode of acute HF. Such patients might be safely discharged.
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| 40. |
- Kinnunen, Juho, et al.
(författare)
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Factors affecting effluent quality in on-site wastewater treatment systems in the cold climates of Finland and Sweden
- 2023
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Ingår i: Journal of Cleaner Production. - : Elsevier. - 0959-6526 .- 1879-1786. ; 404
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Tidskriftsartikel (refereegranskat)abstract
- Decentralized sanitation facilities serving single households to small communities can offer a more flexible way of improving global sanitation. In Nordic countries, on-site wastewater treatment systems (OWTS) are mostly used in sparsely populated areas and are regulated for the removal of phosphorus (Tot-P), organic matter (biological oxygen demand, (BOD7)) and total nitrogen (Tot-N). However, available reports, albeit commonly using small data sets, call attention to often low and variable treatment performance. In this work, a statistical analysis of 1301 samples from 395 units reported in 10 studies from Finland and Sweden was conducted. The objective was to increase knowledge regarding the effluent water quality of OWTS and their compliance with regulations. In addition, the goal was to identify possible factors affecting contaminant concentrations. Overall, compliance with base-level BOD7 removal requirements was met by most units while 10% of units exceeded Tot-N and >25% exceeded Tot-P limits. Non-compliance rates were high, especially for Tot-P, considering the studied data set was composed mostly of semi-new units (median 3.8 years). Neither weather (air temperature, precipitation, and snowmelt), age or load factors were found to strongly explain the data variability in either soil-based systems (SBS) or package plants (PP). Tot-N and Tot-P effluent concentrations of the two systems were not significantly different. Effluent BOD7 concentrations were lower in SBS (median 3.0 mg/L) when compared to PP (median 7.7 mg/L). Units with P-sorbing filters (median 0.69 mg/L) and chemical precipitation (median 1.54 mg/L), presented lower Tot-P effluent concentrations in contrast to traditional sand filters (median 4.0 mg/L). The biological process used in PP had a measurable effect on effluent BOD7 concentrations, with attached growth units presenting higher values (median 8.1 mg/L) than suspended growth units (median 7.1 mg/L). Although the data set utilized was large, its analysis revealed intrinsic bias in data collection, especially regarding system types and sampling seasonality with colder winter periods sampling underrepresented. There is a need for data collection to improve to allow conclusive treatment efficiency assessment including possible effects of climate and process-related factors.
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