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Funder: Knut and Alice Wallenberg Foundation; Id: http://dx.doi.org/10.13039/501100004063, Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 =0.29 μM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol- and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins. publishedVersion

The aim was to clarify the role of vimentin, an intermediate filament protein abundantly expressed in activated macrophages and foam cells, in macrophages during atherogenesis. Global gene expression, lipid uptake, ROS, and inflammation were analyzed in bone-marrow derived macrophages from vimentin-deficient (Vim-/-) and wild-type (Vim+/+) mice. Atherosclerosis was induced in Ldlr-/- mice transplanted with Vim-/- and Vim+/+ bone marrow, and in Vim-/- and Vim+/+ mice injected with a PCSK9 gain-of-function virus. The mice were fed an atherogenic diet for 12-15 weeks. We observed impaired uptake of native LDL but increased uptake of oxLDL in Vim-/- macrophages. FACS analysis revealed increased surface expression of the scavenger receptor CD36 on Vim-/- macrophages. Vim-/- macrophages also displayed increased markers of oxidative stress, activity of the transcription factor NF-κB, secretion of proinflammatory cytokines and GLUT1-mediated glucose uptake. Vim-/- mice displayed decreased atherogenesis despite increased vascular inflammation and increased CD36 expression on macrophages in two mouse models of atherosclerosis. We demonstrate that vimentin has a strong suppressive effect on oxidative stress and that Vim-/- mice display increased vascular inflammation with increased CD36 expression on macrophages despite decreased subendothelial lipid accumulation. Thus, vimentin has a key role in regulating inflammation in macrophages during atherogenesis. This work was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, and the Sahlgrenska University Hospital ALF research grants. Sí

Lactoferrin (LF), a major protein present in milk, mucosal secretions and secondary granules of neutrophils, has been suggested to participate in host defense at mucosal surfaces and to mediate anti-inflammatory activities. A pepsin-derived fragment of LF has been shown to contain the antibacterial domain of the protein. Despite this proposed important dual capacity of LF at the mucosal membranes there are few in vivo studies to support these effects. Our aim was to investigate if anti-infectious or anti-inflammatory activities on mucosal surfaces could be mediated by perorally given LF or synthetic fragments of the antibacterial region of LF in experimental animal models, to gain insight into how LF mediates the anti-inflammatory activities in vitro, and to define the sequence in the antibacterial region of the LF molecule responsible for the antimicrobial activity. Experimental mouse models of urinary tract infection (UTI) induced by E.coli O6K5 and dextran sulphate (DX)-induced acute colitis were used. The number of E.coli present in the urinary tract and the urinary and systemic inflammatory response (urinary leukocytes, urinary and systemic IL-6 levels) in mice with UTI were reduced by the LF treatment compared to the control group. Mice treated with LF peptide fragments (HLD1 and 2) also showed reduced numbers of bacteria in the kidneys. The perorally given LF was found to pass over to the circulation and urine. HLD2 mediated significant bactericidal activity against E.coli when tested in vitro in mouse urine. The damaging effects induced by DX exposure (presence of blood in the faeces, colon shortening, IL-1b serum levels, crypt score) were delayed or reduced in mice treated with LF or the peptides. The number of inflammatory cells present in the colon after 7 days of DX exposure (F4/80 macrophages, CD4- and TNF-a- positive cells) was lower in the LF treated group compared to the control. LF also reduced shortening of the colon when given orally to animals with an already established inflammatory response as induced by two days of DX exposure. LF was shown to down-regulate the secretion of TNF-a, IL-1b, IL-6, IL-8 and IL-10 in monocytic cell lines (THP-1, Mono Mac 6) stimulated with LPS. The down regulation of the cytokines was reflected at the transcriptional level. Thus LPS-induced TNF-a-, IL-1b-, IL-6-, and IL-8 mRNA as shown by reversed transcription PCR were reduced as well. The known binding of LF to LPS could not explain the reduced cytokine mRNA expression and protein secretion since the effects were observed also when LF was added 30 min after the LPS to the cell assay. In addition, also IL-1b induced IL-6 secretion was down-regulated in the presence of LF. Moreover, LF was detected by immunocytochemistry in the cell nucleoli already after 30 min of incubation with the THP-1 cells and found by electromobility shift assay to decrease the binding of nuclear factor (NF)-kB to the TNF-a promoter. The antimicrobial activity against E.coli, S.aureus and C.albicans of synthetic peptides homologous to the surface exposed a-helix and b-sheet region from the N-terminal end of human LF showed that a short region comprising 12-15 a.a. corresponding to the major part of the helix region were optimal for the killing activity against all three microorganisms. In addition certain amino acids such as cystein, and hydrophobic and positively charged amino acids in the 12-amino acid long peptide were found to be important for the expression of antimicrobial activity. In summary, orally given LF can reduce infection and inflammation in a remote site such as the urinary tract, and mediates anti-inflammatory activities in the colon. This dual effect may partly reside in the antimicrobial region of the LF molecule, since synthetic fragments also provide similar activities. One possibly important mechanism of its anti-inflammatory effects is through the ability to down-regulate cytokine production via interference with the transcription factor NF-kB. The anti-infectious and anti-inflammatory activities of LF on mucosal surfaces is being utilized by the suckling child which obtains large amounts of LF via the maternal milk. However, therapeutic use of LF or its fragments may be possible in other fields of application.