Stefanie Frank

Targeting Connexin43 Expression Accelerates the Rate of Wound Repair

The repair of tissue damage is a key survival process in all organisms and involves the coordinated activation of several cell types. Cell-cell communication is clearly fundamental to this process, and a great deal is known about extracellular communication within the wound site via cytokines. Here we show that direct cell-cell communication through connexin 43 (Cx43) gap junction channels also plays a major role in the wound healing process. In two different wound healing models, incisional and excisional skin lesions, we show that a single topical application of Cx43 antisense gel brings about a transient downregulation of Cx43 protein levels, and this results in a dramatic increase in the rate of wound closure. Cx43 knockdown reduces inflammation, seen both macroscopically, as a reduction in swelling, redness, and wound gape, and microscopically, as a significant decrease in neutrophil numbers in the tissue around the wound. One long-term consequence of the improved rate of healing is a significant reduction in the extent of granulation tissue deposition and the subsequent formation of a smaller, less distorted, scar. This approach is likely to have widespread therapeutic applications in other injured tissues and opens up new avenues of research into improving the wound healing process.

Dynamic changes in connexin expression correlate with key events in the wound healing process

Wound healing is a complex process requiring communication for the precise co‐ordination of different cell types. The role of extracellular communication through growth factors in the wound healing process has been extensively documented, but the role of direct intercellular communication via gap junctions has scarcely been investigated. We have examined the dynamics of gap junction protein (Connexins 26, 30, 31.1 and 43) expression in the murine epidermis and dermis during wound healing, and we show that connexin expression is extremely plastic between 6 hours and 12 days post‐wounding. The immediate response (6 h) to wounding is to downregulate all connexins in the epidermis, but thereafter the expression profile of each connexin changes dramatically. Here, we correlate the changing patterns of connexin expression with key events in the wound healing process.

Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.

Bacterial microcompartments moving into a synthetic biological world

Bacterial microcompartments are proteinaceous organelles that are found in a broad range of bacteria. They are composed of an outer protein shell that encases a specific metabolic process. Examples include the carboxysome, which houses enzymes associated with carbon fixation, and the propanediol metabolosome, which contains enzymes linked with the catabolism of propanediol to propionic acid. In this article the molecular structure of bacterial microcompartments is examined and the potential to engineer these intriguing organelles for biotechnological applications is explored.

Structure of a trimeric bacterial microcompartment shell protein, EtuB, associated with ethanol utilization in Clostridium kluyveri.

It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins [EtuA (ethanol utilization shell protein A) and EtuB] are found encoded on the genome clustered with the genes for ethanol utilization. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12)

It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification.

Employing bacterial microcompartment technology to engineer a shell-free enzyme-aggregate for enhanced 1,2-propanediol production in Escherichia coli

Bacterial microcompartments (BMCs) enhance the breakdown of metabolites such as 1,2-propanediol (1,2-PD) to propionic acid. The encapsulation of proteins within the BMC is mediated by the presence of targeting sequences. In an attempt to redesign the Pdu BMC into a 1,2-PD synthesising factory using glycerol as the starting material we added N-terminal targeting peptides to glycerol dehydrogenase, dihydroxyacetone kinase, methylglyoxal synthase and 1,2-propanediol oxidoreductase to allow their inclusion into an empty BMC. 1,2-PD producing strains containing the fused enzymes exhibit a 245% increase in product formation in comparison to un-tagged enzymes, irrespective of the presence of BMCs. Tagging of enzymes with targeting peptides results in the formation of dense protein aggregates within the cell that are shown by immuno-labelling to contain the vast majority of tagged proteins. It can therefore be concluded that these protein inclusions are metabolically active and facilitate the significant increase in product formation.

Elucidation of Substrate Specificity in the Cobalamin (Vitamin B12) Biosynthetic Methyltransferases STRUCTURE AND FUNCTION OF THE C20 METHYLTRANSFERASE (CbiL) FROM METHANOTHERMOBACTER THERMAUTOTROPHICUS

Ring contraction during cobalamin (vitamin B12) biosynthesis requires a seemingly futile methylation of the C20 position of the tetrapyrrole framework. Along the anaerobic route, this reaction is catalyzed by CbiL, which transfers a methyl group from S-adenosyl-l-methionine to cobalt factor II to generate cobalt factor III. CbiL belongs to the class III methyltransferases and displays similarity to other cobalamin biosynthetic methyltransferases that are responsible for the regiospecific methylation of a number of positions on the tetrapyrrole molecular canvas. In an attempt to understand how CbiL selectively methylates the C20 position, a detailed structure function analysis of the enzyme has been undertaken. In this paper, we demonstrate that the enzyme methylates the C20 position, that its preferred substrate is cobalt factor II, and that the metal ion does not undergo any oxidation change during the course of the reaction. The enzyme was crystallized, and its structure was determined by x-ray crystallography, revealing that the 26-kDa protein has a similar overall topology to other class III enzymes. This helped in the identification of some key amino acid residues (Asp104, Lys176, and Tyr220). Analysis of mutant variants of these groups has allowed us to suggest potential roles that these side chains may play in substrate binding and catalysis. EPR analysis of binary and ternary complexes indicate that the protein donates a fifth ligand to the cobalt ion via a gated mechanism to prevent transfer of the methyl group to water. The chemical logic underpinning the methylation is discussed.

Anaerobic synthesis of vitamin B12: characterization of the early steps in the pathway

The anaerobic biosynthesis of vitamin B12 is slowly being unravelled. Recent work has shown that the first committed step along the anaerobic route involves the sirohydrochlorin (chelation of cobalt into factor II). The following enzyme in the pathway, CbiL, methylates cobalt-factor II to give cobalt-factor III. Recent progress on the molecular characterization of this enzyme has given a greater insight into its mode of action and specificity. Structural studies are being used to provide insights into how aspects of this highly complex biosynthetic pathway may have evolved. Between cobalt-factor III and cobyrinic acid, only one further intermediate has been identified. A combination of molecular genetics, recombinant DNA technology and bioorganic chemistry has led to some recent advances in assigning functions to the enzymes of the anaerobic pathway.