Tiina A. Salminen

Heterotrimerization of Heat-Shock Factors 1 and 2 Provides a Transcriptional Switch in Response to Distinct Stimuli

Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.

Functional Characterization of Gne (UDP-N-Acetylglucosamine- 4-Epimerase), Wzz (Chain Length Determinant), and Wzy (O-Antigen Polymerase) of Yersinia enterocolitica Serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.

Crystal structure of the human vascular adhesion protein-1 : Unique structural features with functional implications

The expression of human vascular adhesion protein‐1 (hVAP‐1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X‐ray structure of hVAP‐1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane‐bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP‐1, especially regarding the role of hVAP‐1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N‐glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent‐exposed RGD motif at the entrance to each active site in hVAP‐1 suggests that it may have a functional role.

Operon flv4-flv2 Provides Cyanobacterial Photosystem II with Flexibility of Electron Transfer

This work shows that the flv4-flv2 operon provides many β-cyanobacteria with a so far unknown photoprotection mechanism that evolved in parallel with oxygen-evolving photosystem II Synechocystis sp PCC 6803 has four genes encoding flavodiiron proteins (FDPs; Flv1 to Flv4). Here, we investigated the flv4-flv2 operon encoding the Flv4, Sll0218, and Flv2 proteins, which are strongly expressed under low inorganic carbon conditions (i.e., air level of CO2) but become repressed at elevated CO2 conditions. Different from FDP homodimers in anaerobic microbes, Synechocystis Flv2 and Flv4 form a heterodimer. It is located in cytoplasm but also has a high affinity to membrane in the presence of cations. Sll0218, on the contrary, resides in the thylakoid membrane in association with a high molecular mass protein complex. Sll0218 operates partially independently of Flv2/Flv4. It stabilizes the photosystem II (PSII) dimers, and according to biophysical measurements opens up a novel electron transfer pathway to the Flv2/Flv4 heterodimer from PSII. Constructed homology models suggest efficient electron transfer in heterodimeric Flv2/Flv4. It is suggested that Flv2/Flv4 binds to thylakoids in light, mediates electron transfer from PSII, and concomitantly regulates the association of phycobilisomes with PSII. The function of the flv4-flv2 operon provides many β-cyanobacteria with a so far unknown photoprotection mechanism that evolved in parallel with oxygen-evolving PSII.

Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.

Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the ⁶⁸Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Evolutionary History of the Non-Specific Lipid Transfer Proteins

The non-specific lipid transfer proteins (nsLTPs) are small, basic proteins characterized by a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers. Most nsLTPs are synthesized with an N-terminal signal peptide that localizes the protein to the apoplastic space. The nsLTPs have only been identified in seed plants, where they are encoded by large gene families. We have initiated an analysis of the evolutionary history of the nsLTP family using genomic and EST information from non-seed land plants and green algae to determine: (1) when the nsLTP family arose, (2) how often new nsLTP subfamilies have been created, and (3) how subfamilies differ in their patterns of expansion and loss in different plant lineages. In this study, we searched sequence databases and found that genes and transcripts encoding nsLTPs are abundant in liverworts, mosses, and all other investigated land plants, but not present in any algae. The tertiary structures of representative liverwort and moss nsLTPs were further studied with homology modeling. The results indicate that the nsLTP family has evolved after plants conquered land. Only two of the four major subfamilies of nsLTPs found in flowering plants are present in mosses and liverworts. The additional subfamilies have arisen later, during land plant evolution. In this report, we also introduce a modified nsLTP classification system.

Human Siglec-10 can bind to vascular adhesion protein-1 and serves as its substrate

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10-VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.

A peptide inhibitor of vascular adhesion protein-1 (VAP-1) blocks leukocyte–endothelium interactions under shear stress

Vascular adhesion protein‐1 (VAP‐1) is an endothelial adhesion molecule mediating leukocyte interactions with blood vessels during leukocyte extravasation. Molecularly VAP‐1 is a cell‐surface‐expressed ecto‐enzyme belonging to the group of semicarbazide‐sensitive amine oxidases (SSAO; EC 2.4.6.3), which deaminate primary amines. Here we asked whether peptides displaying a suitable free amine group could be a substrate or inhibitor of SSAO and thus regulate VAP‐1‐mediated leukocyte adhesion. On the basis of a molecular model of VAP‐1, we designed synthetic peptides that fit to the substrate channel of VAP‐1. One of these lysine‐containing peptides effectively inhibits VAP‐1‐dependent lymphocyte rolling and firm adhesion to primary endothelial cells under physiologically relevant shear conditions. The same peptide inhibits the SSAO activity of endothelial and recombinant VAP‐1 in a selective and long‐lasting manner. We also show that all enzymatically active VAP‐1 is displayed on the cell surface. Our results suggest that, in addition to soluble amines, specific cell‐surface‐bound molecules containing free NH2 groups in a suitable position may modulate the enzymaticactivity of SSAO. Moreover, the inhibitory peptide diminishes leukocyte interactions with endothelial cells under conditions of shear, and thus it may be useful to treat inflammatory conditions.

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 Å resolution

The structure of E.coli soluble inorganic pyrophosphatase has been refined at 2.7 A resolution to an R-factor of 20.9%. The overall fold of the molecule is essentially the same as yeast pyrophosphatase, except that yeast pyrophosphatase is longer at both the N- and C-termini. Escherichia coli pyrophosphatase is a mixed alpha + beta protein with a complicated topology. The active site cavity, which is also very similar to the yeast enzyme, is formed by seven beta-strands and an alpha-helix and has a rather asymmetric distribution of charged residues. Our structure-based alignment extends and improves upon earlier sequence alignment studies; it shows that probably no more than 14, not 15-17 charged and polar residues are part of the conserved enzyme mechanism of pyrophosphatases. Six of these conserved residues, at the bottom of the active site cavity, form a tight group centred on Asp70 and probably bind the two essential Mg2+ ions. The others, more spreadout and more positively charged, presumably bind substrate. Escherichia coli pyrophosphatase has an extra aspartate residue in the active site cavity, which may explain why the two enzymes bind divalent cation differently. Based on the structure, we have identified a sequence motif that seems to occur only in soluble inorganic pyrophosphatases.