Recurrence Patterns after Radiotherapy for Glioblastoma with [(11)C]methionine Positron Emission Tomography-Guided Irradiation for Target Volume Optimization.

11C methionine (11C-MET) is increasingly being used in addition to contrast-enhanced MRI to plan for radiotherapy of patients with glioblastomas. This study aimed to assess the recurrence pattern quantitatively. Glioblastoma patients undergoing 11C-MET PET examination before primary radiotherapy from 2018 to 2023 were included in the analysis. A clinical target volume was manually created and fused with MRI-based gross tumor volumes and MET PET-based biological target volume. The recurrence was noted as an area of contrast enhancement on the first MRI scan, which showed progression. The recurrent tumor was identified on the radiological MR images in terms of recurrent tumor volume, and recurrences were classified as central, in-field, marginal, or ex-field tumors. We then compared the MET-PET-defined biological target volume with the MRI-defined recurrent tumor volume regarding spatial overlap (the Dice coefficient) and the Hausdorff distance. Most recurrences occurred locally within the primary tumor area (64.8%). The mean Hausdorff distance was 39.4 mm (SD 32.25), and the mean Dice coefficient was 0.30 (SD 0.22). In patients with glioblastoma, the analysis of the recurrence pattern has been mainly based on FET-PET. Our study confirms that the recurrence pattern after gross tumor volume-based treatment contoured by MET-PET is consistent with the FET-PET-based treatment described in the literature.

Sortase-Mediated Site-Specific Conjugation and 89Zr-Radiolabeling of Designed Ankyrin Repeat Proteins for PET.

Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/neu receptor, was site-specifically modified with enzymatic methods and 89Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and 89Zr-radiolabeling produced a novel 89ZrDFO-G3-DARPin radiotracer that can detect HER2/neu-positive tumors. The triglycine probe, DFO-Gly3 (1), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give 89ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/neu-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of 89ZrDFO-G3-DARPin were measured in vivo by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. 89ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g (n = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, n = 5, P-value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of 89ZrDFO-G3-DARPin in HER2/neu positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of 89ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.

Direct interaction of actin filaments with F-BAR protein pacsin2.

Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.

Protein-peptide complex crystallization: a case study on the ERK2 mitogen-activated protein kinase.

Linear motifs normally bind with only medium binding affinity (Kd of ∼0.1-10 µM) to shallow protein-interaction surfaces on their binding partners. The crystallization of proteins in complex with linear motif-containing peptides is often challenging because the energy gained upon crystal packing between symmetry mates in the crystal may be on a par with the binding energy of the protein-peptide complex. Furthermore, for extracellular signal-regulated kinase 2 (ERK2) the protein-peptide docking surface is comprised of a small hydrophobic surface patch that is often engaged in the crystal packing of apo ERK2 crystals. Here, a rational surface-engineering approach is presented that involves mutating protein surface residues that are distant from the peptide-binding ERK2 docking groove to alanines. These ERK2 surface mutations decrease the chance of `unwanted' crystal packing of ERK2 and the approach led to the structure determination of ERK2 in complex with new docking peptides. These findings highlight the importance of negative selection in crystal engineering for weakly binding protein-peptide complexes.

Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove.

Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear "docking" motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38α, and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38α. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.

Structural basis of heme binding in the Cu,Zn superoxide dismutase from Haemophilus ducreyi.

The Cu,Zn superoxide dismutase from Haemophilus ducreyi is characterized by the unique ability to bind heme at its dimer interface. Here we report the high-resolution crystal structures of this protein in the heme-loaded (holo) and heme-free (apo) forms. Heme is asymmetrically bound between the two enzyme subunits, where heme iron is coordinated by two histidine residues, His64 and His 124, provided by the two subunits. Moreover, the binding of heme to the protein is ensured by stabilizing contacts between the prosthetic group and a limited number of other residues, most of which are not present in other bacterial enzyme variants. We show that the introduction of only three mutations at the dimer interface of the enzyme from Haemophilus parainfluenzae, a closely related bacterial species, is sufficient to induce heme-binding ability by this enzyme variant. Heme binding does not alter protein activity. Moreover, the binding of the prosthetic group does not induce any significant structural perturbation at the subunit level and requires only limited local structural rearrangements that widen the cleft at the dimer interface and cause a limited shift in the relative orientation between the subunits. The presence of a preformed heme-binding pocket and the significant solvent exposure of the cofactor to the solvent are compatible with the suggested protective role of the enzyme against heme toxicity or with its involvement in heme trafficking in the periplasmic space.

Unique features of the sodC-encoded superoxide dismutase from Mycobacterium tuberculosis, a fully functional copper-containing enzyme lacking zinc in the active site.

The sodC-encoded Mycobacterium tuberculosis superoxide dismutase (SOD) shows high sequence homology to other members of the copper/zinc-containing SOD family. Its three-dimensional structure is reported here, solved by x-ray crystallography at 1.63-A resolution. Metal analyses of the recombinant protein indicate that the native form of the enzyme lacks the zinc ion, which has a very important structural and functional role in all other known enzymes of this class. The absence of zinc within the active site is due to significant rearrangements in the zinc subloop, including deletion or mutation of the metal ligands His115 and His123. Nonetheless, the enzyme has a catalytic rate close to the diffusion limit; and unlike all other copper/zinc-containing SODs devoid of zinc, the geometry of the copper site is pH-independent. The protein shows a novel dimer interface characterized by a long and rigid loop, which confers structural stability to the enzyme. As the survival of bacterial pathogens within their host critically depends on their ability to recruit zinc in highly competitive environments, we propose that the observed structural rearrangements are required to build up a zinc-independent but fully active and stable copper-containing SOD.

Crystallization and phasing of focal adhesion protein 52 from Gallus gallus.

Focal adhesion protein 52 (FAP52) is a multidomain adaptor protein of 448 amino acids characterized as an abundant component of focal adhesions. FAP52 binds to filamin via its N-terminal alpha-helical domain, suggesting a role in linking focal adhesions to the actin-based cytoskeleton. The recombinant protein was crystallized using the hanging-drop vapour-diffusion method, which yielded two crystal forms. Native data were collected from both crystal forms to 2.8 and 2.1 A resolution, respectively. For one of the crystal forms, initial MAD phasing was successfully performed using two data sets from xenon-derivatized crystals. The derivative data sets were collected using softer X-rays of 1.5 and 1.9 A wavelength. Preliminary structural analysis reveals the presence of a dimer in the asymmetric unit.

Archaeal Sm proteins form heptameric and hexameric complexes: crystal structures of the Sm1 and Sm2 proteins from the hyperthermophile Archaeoglobus fulgidus.

Proteins of largely unknown function related to the Sm proteins present in the core domain of eukaryotic small nuclear ribonucleoprotein particles have recently been detected in Archaea. In contrast to eukaryotes, Archaea contain maximally two distinct Sm-related proteins belonging to different subfamilies, we refer to as Sm1 and Sm2. Here we report the crystal structures of the Sm1- and Sm2-type proteins from the hyperthermophilic euryarchaeon Archaeoglobus fulgidus (AF-Sm1 and AF-Sm2) at a resolution of 2.5 and 1.95 A, respectively. While the AF-Sm1 protein forms a heptameric ring structure similar to that found in other archaeal Sm1-type proteins, the AF-Sm2 protein unexpectedly forms a homo-hexamer in the crystals, and, as is evident from the mass spectrometric analysis, also in solution. Both proteins have essentially the same monomer fold and inter-subunit beta-sheet hydrogen bonding giving rise to a similar overall architecture of the doughnut-shaped six and seven-membered rings. In addition, a conserved uracil-binding pocket identified previously in an AF-Sm1/RNA complex, suggests a common RNA-binding mode for the AF-Sm1 and AF-Sm2 proteins, in line with solution studies showing preferential binding to U-rich oligonucleotides for both proteins. Clear differences are however seen in the charge distribution within the two structures. The rough faces of the rings, i.e. the faces not containing the base binding pockets, have opposite charges in the two structures, being predominantly positive in AF-Sm1 and negative in AF-Sm2. Differences in the ionic interactions between subunits provide an explanation for the distinctly different oligomerisation behaviour of the AF-Sm1 and AF-Sm2 proteins and of Sm1- and Sm2-type proteins in general, as well as the stability of their complexes. Implications for the functions of archaeal Sm proteins are being discussed.