Structure of a protein-DNA complex essential for DNA protection in spores of Bacillus species.

The DNA-binding alpha/beta-type small acid-soluble proteins (SASPs) are a major factor in the resistance and long-term survival of spores of Bacillus species by protecting spore DNA against damage due to desiccation, heat, toxic chemicals, enzymes, and UV radiation. We now report the crystal structure at 2.1 A resolution of an alpha/beta-type SASP bound to a 10-bp DNA duplex. In the complex, the alpha/beta-type SASP adopt a helix-turn-helix motif, interact with DNA through minor groove contacts, bind to approximately 6 bp of DNA as a dimer, and the DNA is in an A-B type conformation. The structure of the complex provides important insights into the molecular details of both DNA and alpha/beta-type SASP protection in the complex and thus also in spores.

The motif of SPARC that inhibits DNA synthesis is not a nuclear localization signal.

SPARC (secreted protein acidic and rich in cysteine), although primarily known as a secreted, matricellular protein, has also been identified in urothelial cell nuclei. Many biological activities, including inhibition of cell adhesion and repression of DNA synthesis, have been ascribed to SPARC, but the influence of its intracellular localization on each of these activities is unknown. When exposed by epitope retrieval and nuclear matrix unmasking techniques, endogenous SPARC was found to localize strongly to the nuclei and the nuclear matrix of cultured urothelial cells. Live-cell time-lapse imaging revealed that exogenous fluorescently labeled recombinant (r) SPARC was taken up from medium over a 16 h period and accumulated inside cells. Two variants of rSPARC with alterations in its putative nuclear localization signal (NLS) were generated to investigate the existence and effects of the NLS. These variants demonstrated similar biophysical characteristics as the wild-type protein. Visualization by a variety of techniques, including live-cell imaging, deconvolution microscopy, and cell fractionation, all concurred that exogenous rSPARC was not able to localize to cell nuclei, but instead accumulated as perinuclear clusters. Localization of the rSPARC NLS variants was no different than wild-type, arguing against the presence of an active NLS in rSPARC. Imaging experiments showed that only permeabilized, dead cells avidly took up rSPARC into their nuclei. The rSPARC(no NLS) variant proved ineffective at inhibiting DNA synthesis, whereas the rSPARC(strong NLS) variant was a more potent inhibitor of DNA synthesis than was wild-type rSPARC. The motif of SPARC that inhibits the synthesis of urothelial cell DNA is therefore not a nuclear localization signal, but its manipulation holds therapeutic potential to generate a "Super-SPARC" that can quiesce proliferative tissues.

Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis alpha/beta-type small acid-soluble spore protein and DNA.

An engineered variant of an alpha/beta-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 A resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 87.0, c = 145.4 A, alpha = beta = 90.0, gamma = 120.0 degrees. Diffraction data were 96.6% complete to 2.4 A resolution, with an R(sym) of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.

Translocation of fibroblast growth factor-10 and its receptor into nuclei of human urothelial cells.

Fibroblast growth factor-10 (FGF-10), a mitogen for the epithelial cells lining the lower urinary tract, has been identified inside urothelial cells, despite its acknowledged role as an extracellular signaling ligand. Recombinant (r)FGF-10 was determined by fluorescence microscopy optical sectioning to localize strongly to nuclei inside cultured urothelial cells. To clarify the possible role of a nuclear localization signal (NLS) in this translocation, a variant of rFGF-10 was constructed which lacked this sequence. rFGF-10(no NLS) was found in cytoplasm to a far greater degree than rFGF-10, identifying this motif as a possible NLS. Furthermore, this variant displayed poor or non-existent bioactivity compared to the wild-type protein in triggering mitogenesis in quiescent urothelial cells. The presence of rFGF-10(no NLS) in the nucleus suggested that additional interactions were also responsible for the nuclear accumulation of rFGF-10. The FGF-10 receptor was observed in cell nuclei regardless of the presence or concentration of exogenous rFGF-10 ligand. Co-localization studies between rFGF-10 and the FGF-10 receptor revealed a strong intracellular relationship between the two. This co-localization was seen in nuclei for both rFGF-10 and for rFGF-10(no NLS), although the correlation was weaker for rFGF-10(no NLS). These data show that an NLS-like motif of rFGF-10 is a partial determinant of its intracellular distribution and is necessary for its mitogenic activity. These advancements in the understanding of the activity of FGF-10 present an opportunity to engineer the growth factor as a therapeutic agent for the healing of damaged urothelial tissue.

FGF-10 and its receptor exhibit bidirectional paracrine targeting to urothelial and smooth muscle cells in the lower urinary tract.

Control of the regenerative properties of urothelial tissue would greatly aid the clinician in the management of urinary tract disease and disorders. Fibroblast growth factor 10 (FGF-10) is a mitogen which is particularly promising as a protein therapy for urothelial injury. The spatial synthesis, transport, targeting, and mechanistic pathway of FGF-10 and its receptor were studied in a human urothelial cell culture model and in fixed sections of lower urinary tract tissue. Synthesis of FGF-10 was restricted to mesenchymal fibroblasts, and secreted FGF-10 exhibited paracrine transport to two proximal sites, transitional epithelium and smooth muscle cell bundles, both of which were also the exclusive sites of FGF-10 receptor synthesis. The addition of recombinant FGF-10 to quiescent urothelial cells in vitro was sufficient to stimulate DNA synthesis. This stimulation was through a pathway independent of the epidermal growth factor receptor pathway. Deconvolution, light and transmission electron microscopic studies captured FGF-10 and its receptor in association with the urothelial cell surface, in cytoplasm, and within nuclei, observations that describe the mechanism that transduces the mitogenic signal in these tissues. Localization of the FGF-10 receptor to the superficial urothelial layer is clinically significant because intravesical administration of FGF-10 may provide the clinician a means to control the turnover of transitional epithelium in bladder disorders such as interstitial cystitis.

The C-terminal Ca2+-binding domain of SPARC confers anti-spreading activity to human urothelial cells.

The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .

Effects of carboxy-terminal modifications and pH on binding of a Bacillus subtilis small, acid-soluble spore protein to DNA.

Variants of the wild-type Bacillus subtilis alpha/beta-type small, acid-soluble spore protein (SASP) SspC(wt) were designed to evaluate the contribution of C-terminal residues to these proteins' affinity for DNA. SspC variants lacking one to three C-terminal residues were similar to SspC(wt) in DNA binding, but removal of six C-terminal residues greatly decreased DNA binding. In contrast, a C-terminal extension of three residues increased SspC's affinity for DNA 5- to 10-fold. C-terminal and N-terminal changes that independently caused large increases in SspC-DNA binding affinity were combined and produced an additive effect on DNA binding; the affinity of the resulting variant, SspC(DeltaN11-D13K-C3), for DNA was increased >/==" BORDER="0">20-fold over that of SspC(wt). For most of the SspC variants tested, lowering the pH from 7 to 6 improved DNA binding two- to sixfold, although the opposite effect was observed with variants having additional C-terminal basic residues. In vitro, the binding of SspC(DeltaN11-D13K-C3) to DNA suppressed the formation of cyclobutane-type thymine dimers and promoted the formation of the spore photoproduct upon UV irradiation to the same degree as the binding of SspC(wt). However, B. subtilis spores lacking major alpha/beta-type SASP and overexpressing SspC(DeltaN11-D13K-C3) had a 10-fold-lower viability and far less UV and heat resistance than spores overexpressing SspC(wt). This apparent lack of DNA protection by SspC(DeltaN11-D13K-C3) in vivo is likely due to the twofold-lower level of this protein in spores compared to the level of SspC(wt), perhaps because of effects of SspC(DeltaN11-D13K-C3) on gene expression in the forespore during sporulation. The latter results indicate that only moderately strong binding of alpha/beta-type SASP to DNA is important to balance the potentially conflicting requirements for these proteins in DNA transcription and DNA protection during spore formation, spore dormancy, and spore germination and outgrowth.