Functional assessment of hydrophilic domains of late embryogenesis abundant proteins from distant organisms

Late embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11-amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α-helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H₂O₂), suggesting a protective role in a chaperone-like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress-response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability.     

Isolation and characterization of hemidesmosomes from bovine corneal epithelial cells

The hemidesmosome (HD) is a specialized cell-to-substratum junction of stratified and complex epithelia which is characterized by a cytoplasmic plaque to which intermediate filaments (IFs) are anchored. To identify and characterize HD constituents systematically, we have developed a procedure to isolate and fractionate HDs. When bovine corneal epithelium is peeled off from the extracellular matrix stroma, HDs attached to the basal lamina are left behind, together with tufts of cytokeratin IFs attached to the cytoplasmic HD plaques. After rinsing these residual basal cell elements with EDTA, the HDs could be mechanically detached from the stroma and collected by centrifugation. The fraction obtained was examined biochemically and electron microscopically, showing enrichment of HD structures as well as of a prominent 230-kDa polypeptide, the "pemphigoid antigen" known to be located in the HD plaque. In addition, the HD fraction revealed, besides residual amounts of corneal cytokeratins, major polypeptides of Mr 120, 180, 200, 230, and 480 kDa, of which the first three appeared to be glycoproteins. Using the isolated HDs for immunization, we prepared monoclonal antibodies specific for the 230- and 180-kDa polypeptides, respectively, and showed that both were exclusively located in HDs. This method for isolating HDs and the availability of antibodies to HD proteins will be useful in studies of the molecular organization of HDs and make HD research independent from human autoimmune antibodies.     

Regulation of the Type II Hemidesmosomal Plaque Assembly in Intestinal Epithelial Cells

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.     

The interaction with DNA of wild-type and mutant fushi tarazu homeodomains.

The in vitro DNA binding properties of wild-type and mutant fushi tarazu homeodomains (ftz HD) have been analysed. The DNA binding properties of the ftz HD are very similar to those of the Antp HD. In interference experiments with mutant ftz HDs, close approaches between specific portions of the ftz HD peptide and specific regions of the binding site DNA were mapped. A methylation interference, G7 on the beta strand of BS2, is absent from the interference pattern with a mutant ftz HD [ftz (R43A) HD] in which the Arg43 at the second position of helix III (the recognition helix) is replaced by an Ala. This indicated that Arg43 of the ftz HD is in close proximity to the N7 of G7 of the beta strand of BS2 in the major groove. The methylation and ethylation interference patterns with the ftz (NTD) HD, in which the first six amino acids of the homeodomain were deleted, were extensively altered relative to the ftz HD patterns. Methylation of A11 and G12 of the alpha strand and ethylation of the phosphate of nucleotide A12 of the alpha strand no longer interfere with binding. This indicated that the first six amino acids of the homeodomain of ftz interact with A11 of the alpha strand in the minor groove, the phosphate of the nucleotide A13 on the alpha strand and G12 of the alpha strand in the adjacent major groove of BS2. In a binding study using a change of specificity mutation [ftz (Q50K) HD], in which the Gln50 at the ninth position of the third helix is exchanged for a Lys (as in the bicoid HD), and variant binding sites, we concluded that position 50 of the ftz HD and the ftz (Q50K) HD peptides interacts with base pairs at positions 6 and 7 of BS2. These three points of contact allowed us to propose a crude orientation of the ftz HD within the protein-DNA complex. We find that the ftz HD and the Antp HD peptides contact DNA in a similar way.     

Initiation of the microgene polymerization reaction with non-repetitive homo-duplexes

Microgene Polymerization Reaction (MPR) is used as an experimental system to artificially simulate evolution of short, non-repetitive homo-duplex DNA into multiply-repetitive products that can code for functional proteins. Blunt-end ligation by DNA polymerase is crucial in expansion of homo-duplexes (HDs) into head-to-tail multiple repeats in MPR. The propagation mechanism is known, but formation of the initial doublet (ID) by juxtaposing two HDs and polymerization through the gap has been ambiguous. Initiation events with pairs of HDs using Real-Time PCR were more frequent at higher HD concentrations and slightly below the melting temperature. A process molecularity of about 3.1, calculated from the amplification efficiency and the difference in PCR cycles at which propagation was detected at varying HD concentrations, led to a simple mechanism for ID formation: the gap between two HDs is bridged by a third. Considering thermodynamic aspects of the presumed intermediate “nucleation complex” can predict relative propensity for the process with other HDs.     

Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes.

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.     

Using the recognition code to swap homeodomain target specificity in cell culture

The homeodomain (HD) is a 60 amino acid-long DNA-binding domain. A large fraction of HDs binds with high affinity sequences containing the 5′-TAAT-3′ core motif. However, NK-2 class HDs recognizes sequences containing the 5′-CAAG-3′ core motif. By using a cell transfection approach, here we show that modification of residues located in the N-terminal arm (at positions 6, 7 and 8) and in the recognition helix (at position 54) is enough to swap the “in vivo” binding specificity of TTF-1 HD (which is a member of the NK-2 class HD) from 5′-CAAG-3′ to 5′-TAAT-3′-containing targets. The role of residue at position 54 is also supported by data obtained with the HD of the Drosophila engrailed protein. These data support the notion that DNA-binding specificity “in vivo” is dictated by few critical residues.     

The sequence specificity of homeodomain-DNA interaction

The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Using deletion constructs expressed as fusion proteins in E. coli, we localized this activity to the conserved homeodomain (HD). The binding site consensus, TCAATTAAAT, is found in clusters in the engrailed regulatory region. Weak binding of the En HD to one copy of a synthetic consensus is enhanced by adjacent copies. The distantly related HD encoded by fushi tarazu binds to the same sites as the En HD, but differs in its preference for related sites. Both HDs bind a second type of sequence, a repeat of TAA. The similarity in sequence specificity of En and Ftz HDs suggests that, within families of DNA binding proteins, close relatives will exhibit similar specificities. Competition among related regulatory proteins might govern which protein occupies a given binding site and consequently determine the ultimate effect of cis-acting regulatory sites.     

Engineering the loop region of a homeodomain-leucine zipper protein promotes efficient binding to a monomeric DNA binding site

Plant homeodomain-leucine zipper (HD-Zip) proteins, unlike many animal homeodomains (HDs), are unable to bind DNA as monomers. To investigate the molecular basis of their different behavior, we have constructed chimeras between the HD of the sunflower HD-Zip protein Hahb-4 and that of Drosophila engrailed (EN). Analysis of the interaction of these proteins with the pseudopalindromic Hahb-4 binding site and the monomeric EN binding site suggests that the loop located between helix I and helix II (amino acids 21-28) of EN is enough to confer efficient DNA binding activity to the Hahb-4 HD. Accordingly, the combined mutation of residues 24 and 25 of Hahb-4 to those present in EN (S24R/R25Y) originated an HD able to interact with the EN binding site, while single mutations were ineffective. We have also determined that a protein with the leucine zipper and helix III of Hahb-4 fused to the rest of the EN HD binds to the Hahb-4 pseudopalindomic binding site with increased affinity and shows extended contacts with DNA respective to Hahb-4. We conclude that the loop located between helix I and helix II of the HD must be regarded as one of the segments that contribute to the present-day diversity in the properties of different HDs.