Disseksjon av vitellogenins molekylære egenskaper : et protein med funksjoner i grenseflaten mellom sosial adferd og aldring

Vitellogenin is a central honey bee (Apis mellifera) life-history regulator. This thesis represents an initiative to study this protein, which affects aging and task-division of the bees, at the molecular level. I have used, among others, molecular modeling, nuclear magnetic resonance spectroscopy and surface plasmon resonance techniques to understand the structure and properties of vitellogenin. In addition to the three experimental papers of this thesis, vitellogenin is discussed from the molecular viewpoint in two invited publications (Papers II-III). The studies have resulted in more detailed understanding of the structural organization of the protein and its modifications: the novel findings include the cleavage of honey bee vitellogenin at a phosphorylated polyserine linker site, and the ability of the protein to bind to membranes and to interact with Escherichia coli. Thus, this vitellogenin study exceeds its primary molecular scope, and provides a new perspective on the protein as a membrane-active factor in bee physiology. Since little is known of the detailed molecular properties of insect vitellogenins in general and honey bee vitellogenin in particular, the thesis also contributes towards expanding vitellogenin molecular biology beyond the more studied vertebrate vitellogenins.Vitellogenin er et sentralt protein i reguleringen av livsutviklingen hos honningbie (Apis mellifera). Dette proteinet påvirker aldring og oppgavefordeling hos bier, og avhandlingen er en studie av proteinet på molekylært nivå. Jeg har blant annet brukt molekylær modellering, kjernemagnetisk resonans spektroskopi og en teknikk for å måle overflateplasmonresonans (surface plasmon resonance) for å forstå strukturen og egenskapene til vitellogenin. I tillegg til de tre eksperimentelle artiklene i denne avhandlingen er vitellogenin diskutert fra et molekylært synspunkt i to inviterte publikasjoner (Papers II-III). Studiene har resultert i en økt forståelse av den strukturelle oppbygning av proteinet og dets modifikasjoner. Blant viktige funn i avhandlingen kan det nevnes at vitellogenin blir spaltet på et fosforylert polyserin-linker sted, at det binder seg til membraner og at det interagerer med Escherichia coli. Denne studiens omfang dekker dermed mer enn det primært molekylære, og gir nye perspektiv på proteiner som en membran-aktiv faktor i biefysiologi. Detaljert kjennskap om de molekylære egenskapene til vitellogenin hos insekter, og hos honningbie spesielt, er mangelfull, og denne avhandlingen gir derfor et bidrag til å utvide kunnskapen om molekylærbiologien til vitellogenin utover de mer studerte vitellogeninene hos vertebrater

Insights into the Evolution of the CSP Gene Family through the Integration of Evolutionary Analysis and Comparative Protein Modeling

Peer reviewe

From Sustainability-as-usual to Sustainability Excellence in Local Bioenergy Business

Bioenergy business operators can significantly contribute to the sustainability of bioenergy systems. While research has addressed the maturity of corporate responsibility for sustainability, the maturity levels of bioenergy business have not been determined. The objectives of this research were to characterise the maturity levels of bioenergy corporate responsibility for sustainability and outline an approach by which companies can operate at the most mature sustainability excellence level. Literature, three workshops attended by bioenergy experts and a case study on biobutanol production in Brazil were used to develop the maturity model and approach. The results characterise the profitability, acceptability, and sustainability orientation maturity levels through sustainability questions and methods, and list the components of a systemic, holistic approach. Although the shift of business mindset from sustainability-as-usual to sustainability excellence is challenging, a systemic approach is necessary to broadly identify sustainability questions and a multitude of methods by which they can be answered

Development of an RNA Interference Tool, Characterization of Its Target, and an Ecological Test of Caste Differentiation in the Eusocial Wasp Polistes

Recent advancements in genomics provide new tools for evolutionary ecological research. The paper wasp genus Polistes is a model for social insect evolution and behavioral ecology. We developed RNA interference (RNAi)-mediated gene silencing to explore proposed connections between expression of hexameric storage proteins and worker vs. gyne (potential future foundress) castes in naturally-founded colonies of P. metricus. We extended four fragments of putative hexamerin-encoding P. metricus transcripts acquired from a previous study and fully sequenced a gene that encodes Hexamerin 2, one of two proposed hexameric storage proteins of P. metricus. MALDI-TOF/TOF, LC-MSMS, deglycosylation, and detection of phosphorylation assays showed that the two putative hexamerins diverge in peptide sequence and biochemistry. We targeted the hexamerin 2 gene in 5th (last)-instar larvae by feeding RNAi-inducing double-stranded hexamerin 2 RNA directly to larvae in naturally-founded colonies in the field. Larval development and adult traits were not significantly altered in hexamerin 2 knockdowns, but there were suggestive trends toward increased developmental time and less developed ovaries, which are gyne characteristics. By demonstrating how data acquisition from 454/Roche pyrosequencing can be combined with biochemical and proteomics assays and how RNAi can be deployed successfully in field experiments on Polistes, our results pave the way for functional genomic research that can contribute significantly to learning the interactions of environment, development, and the roles they play in paper wasp evolution and behavioral ecology

Differences in the size of the CSP binding pockets can reflect ligand differences.

<p>The N-terminus is indicated. The 34 binding-residues are shown as sticks. Certain binding-residues of interest are indicated in each CSP. (A) Residues within 5 Å of the ligands in the ligand-bound <i>M. brassicae</i> structure (PDB-ID: 1N8V) were considered as binding-residues. The bromo-dodecanol ligands are visualized inside the pocket. (B) The model of CSP1 shown here represents a binding pocket of a non-ligand bound typical CSP protein. (C) The binding pocket of CSP5 is likely enlarged due to the lack of helix 6 and mutations that reduce the size of the binding-residues (A44, Q54, A58 V67 and V36). (D) The binding pocket of CSP7 model is crowded by large binding-residues. For example, F50 and W28 are larger than the corresponding amino acid residues in other, typical CSPs.</p

Size distribution of the binding pocket residues.

<p>Size distribution of the binding pocket residues.</p

Ant-specific CSPs share similarities with CSP7, and positive selection in them is concentrated on the surface.

<p>(A) Left, molecular model of CSP7 shows a “crown” of charged residues (circled). The crown is formed by the positive loop between helices 3 and 4, and by the negative charge between helices 5 and 6. The ant-specific proteins (right) all have this crown. Positively charged residues (K, R) are shown in blue and negatively charged (D, E) in red. (B) The ten residues under positive selection (shown as sticks on the peptide backbone) mostly map on the surface. L87 and L91 are the only binding residues under positive selection. K58 and A66 are located near the “crown” and have various combinations of positive charge and hydrophobicity in the ant-specific CSPs. The N and C termini are indicated.</p

Surface charge variation in orthologous ant CSPs between the seven ant species.

<p>Surface charge variation in orthologous ant CSPs between the seven ant species.</p

Groups of ant CSPs based on their phylogeny and structural models.

<p>All ants share seven orthologous CSPs (CSP1-7; the uppermost row of models), of which CSP7 or a protein similar to that has given rise to the ant-specific expansions (representative proteins on the middle row). The largest currently known ant-expansion is found in <i>S. invicta</i> (the lowest row). In the orthologs, CSP1-4 can be grouped together based on their evenly speckled surface charges and similarities in their binding pocket (“Typical CSPs”; green). CSP5 (grey) is conserved across arthropods, and is one of the oldest CSPs. It differs from the other orthologs by having five instead of six helices, a reduced charge on the surface and by changes to its binding pocket. CSP6 and CSP7 are grouped together (purple) due to mutations in their binding pocket that are likely to reduce the size of the pocket cavity. Examples of the ant-specific CSP expansion are shown; <i>Atta cephalotes</i> CSP8, <i>Pogonomyrmex barbatus</i> CSP10, <i>Camponotus floridanus</i> CSP11, <i>Acromyrmex echinatior</i> CSP14 and <i>Solenopsis invicta</i> CSP17. In the models, negatively charged amino acids (E, D) are shown in red and positively charged amino acids (K, R) in blue. C-termini are marked by an arrow. The <i>S. invicta</i> expansion proteins, together with CSP5, are one helix shorter than the other CSPs in their C-terminus, which is depicted by the altered location of the arrow in these models.</p