Sclerostin antibody enhances implant osseointegration in bone with Col1a1 mutation.

We evaluated the potential of sclerostin antibody (SclAb) therapy to enhance osseointegration of dental and orthopaedic implants in a mouse model (Brtl/+) mimicking moderate to severe Osteogenesis Imperfecta (OI). To address the challenges in achieving stable implant integration in compromised bone conditions, our aim was to determine the effectiveness of sclerostin antibody (SclAb) at improving bone-to-implant contact and implant fixation strength. Utilizing a combination of micro-computed tomography, mechanical push-in testing, immunohistochemistry, and Western blot analysis, we observed that SclAb treatment significantly enhances bone volume fraction (BV/TV) and bone-implant contact (BIC) in Brtl/+ mice, suggesting a normalization of bone structure toward WT levels. Despite variations in implant survival rates between the maxilla and tibia, SclAb treatment consistently improved implant stability and resistance to mechanical forces, highlighting its potential to overcome the inherent challenges of OI in dental and orthopaedic implant integration. These results suggest that SclAb could be a valuable therapeutic approach for enhancing implant success in compromised bone conditions.

Characterizing the protein-protein interaction between MDM2 and 14-3-3σ; proof of concept for small molecule stabilization.

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

Binding of l-kynurenine to X. campestris tryptophan 2,3-dioxygenase.

The kynurenine pathway is the major route of tryptophan metabolism. The first step of this pathway is catalysed by one of two heme-dependent dioxygenase enzymes - tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) - leading initially to the formation of N-formylkynurenine (NFK). In this paper, we present a crystal structure of a bacterial TDO from X. campestris in complex with l-kynurenine, the hydrolysed product of NFK. l-kynurenine is bound at the active site in a similar location to the substrate (l-Trp). Hydrogen bonding interactions with Arg117 and the heme 7-propionate anchor the l-kynurenine molecule into the pocket. A mechanism for the hydrolysis of NFK in the active site is presented.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV =O or FeIV -OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV =O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV=O or FeIV-OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

A Case Study on the Customized Nutrition Intervention for a Patient with Primary Gastrointestinal Non-Hodgkin Lymphoma Underlying Chronic Kidney Disease.

Non-Hodgkin lymphoma comprises 2.1% of the total number of cancers in South Korea. Among those, diffuse large B cell lymphoma (DLBCL) comprises the largest percentage. Nutrition interventions have been highlighted because nutritional status in non-Hodgkin's lymphoma patients has a significant impact on treatment and prognosis, but relevant studies are inadequate. Therefore, the aim of this study was to share the case of a nutrition intervention for a patient with primary gastrointestinal non-Hodgkin lymphoma underlying chronic kidney disease who was comorbid with tumor lysis syndrome, which was a complication of a specific chemotherapy. The subject is a 76-year-old patient who was diagnosed with DLBCL. He had abdominal pain, constipation, and anorexia. After chemotherapy, he experienced the tumor lysis syndrome. The patient's condition was continuously monitored, and various nutrition interventions, such as nutrition counseling and education, provision of therapeutic diet, oral nutritional supplement, change of meal plans, and parenteral nutrition support were attempted. As a result of the nutrition intervention, oral intake was increased from 27% of the energy requirement to 70% and from 23% of the protein requirement to 77%. Despite the various nutrition interventions during the hospitalization, there were no improvements in weight and nutrition-related biochemical parameters or malnutrition. However, it was meaningful in that the patient was managed to prevent worsening and the planned third chemotherapy could be performed. These results can be used as the basis for establishing guidelines for nutritional interventions customized to patients under the same conditions.

Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel.

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Visualizing the protons in a metalloenzyme electron proton transfer pathway.

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation.

Heme peroxidase-Trapping intermediates by cryo neutron crystallography.

By combining the normal practice for X-ray crystallography of collecting diffraction data at 100K with neutron crystallography the structures of cryo-trapped enzyme intermediates have been determined, revealing the positions of the previously hidden hydrogens that are essential to a better understanding of the involved mechanism.

Heme binding to human CLOCK affects interactions with the E-box.

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.

The rise of neutron cryo-crystallography.

The use of boiled-off liquid nitrogen to maintain protein crystals at 100 K during X-ray data collection has become almost universal. Applying this to neutron protein crystallography offers the opportunity to significantly broaden the scope of biochemical problems that can be addressed, although care must be taken in assuming that direct extrapolation to room temperature is always valid. Here, the history to date of neutron protein cryo-crystallography and the particular problems and solutions associated with the mounting and cryocooling of the larger crystals needed for neutron crystallography are reviewed. Finally, the outlook for further cryogenic neutron studies using existing and future neutron instrumentation is discussed.

The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase.

Ascorbate peroxidase (APX) is a class I heme peroxidase. It has two sites for binding of substrates. One is close to the γ-heme edge and is used for oxidation of ascorbate; the other is at the δ-heme edge and is used for binding of aromatic substrates [Gumiero et al., (2010) Arch. Biochem. Biophys. 500, 13-20]. In this work, we have examined the structural factors that control binding at the δ-heme edge by replacement of Ala134 in APX with a proline residue that is more commonly found in other class II and III peroxidases. Kinetic data indicate that replacement of Ala134 by proline has only a small effect on the catalytic mechanism, or the oxidation of ascorbate or guaiacol. Chemical modification with phenylhydrazine indicates that heme accessibility close to the δ-heme edge is only minorly affected by the substitution. We conclude that the A134P mutation alone is not enough to substantially affect the reactivity of APX towards aromatic substrates bound at the δ-heme edge. The data are relevant to the recent application of APX (APEX) in cellular imaging.

Engineering a Lys-Asn isopeptide bond into an immunoglobulin-like protein domain enhances its stability.

The overall stability of globular protein structures is marginal, a balance between large numbers of stabilizing non-covalent interactions and a destabilizing entropic term. Higher stability can be engineered by introduction of disulfide bonds, provided the redox environment is controlled. The discovery of stabilizing isopeptide bond crosslinks, formed spontaneously between lysine and asparagine (or aspartic acid) side chains in certain bacterial cell-surface proteins suggests that such bonds could be introduced by protein engineering as an alternative protein stabilization strategy. We report the first example of an isopeptide bond engineered de novo into an immunoglobulin-like protein, the minor pilin FctB from Streptococcus pyogenes. Four mutations were sufficient; lysine, asparagine and glutamic acid residues were introduced for the bond-forming reaction, with a fourth Val/Phe mutation to help steer the lysine side chain into position. The spontaneously-formed isopeptide bond was confirmed by mass spectrometry and X-ray crystallography, and was shown to increase the thermal stability by 10 °C compared with the wild type protein. This novel method for increasing the stability of IgG-like proteins has potential to be adopted by the field of antibody engineering, which share similar ß-clasp Ig-type domains.

Combining X-ray and neutron crystallography with spectroscopy.

X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H+ ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction.

Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme.

Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)-OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)-OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)-OH in a peroxidase.

Regulation of intracellular heme trafficking revealed by subcellular reporters.

Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

Incidence of isolated dipstick hematuria and its association with the glomerular filtration rate: a cross-sectional study from the Korean National Health and Nutrition Examination Survey V (2010-2012).

PURPOSE: To investigate the incidence of isolated hematuria and its relationship to the glomerular filtration rate (GFR). METHODS: Data from the Korean National Health and Nutrition Examination Survey V were used. A stratified, three-stage, clustered probability design was used to collect representative data on the Korean population. Ultimately, 18,587 participants were included. The incidence of isolated dipstick hematuria and its relationship with the GFR (estimated by the Chronic Kidney Disease Epidemiology Collaboration equation) were evaluated. RESULTS: The analysis showed that 31.8% of the population had isolated hematuria, the incidence of which significantly increased with age (P trend < 0.001). As the severity of hematuria increased, the ratio of GFR < 60 ml/min/1.73 m(2) and 60 ml/min/1.73 m(2) ≤ GFR < 90 ml/min/1.73 m(2) was significantly increased (P trend < 0.001). After adjusting for the confounders, the mean GFR of the grade 3+ (grades 3, 4, 5) hematuria group was significantly reduced compared to that of the negative, grade 1, and grade 2 hematuria groups (with an adjusted mean ± standard error of 94.0 ± 0.8 vs. 97.2 ± 0.3 ml/min/1.73 m(2), P < 0.001). Additionally, the odds ratio of the grade 3+ hematuria group for a GFR < 60 ml/min/1.73 m(2) was significantly increased compared to that of the negative, grade 1, and grade 2 hematuria groups after adjusting for the confounders (adjusted odds ratio 1.468, 95% confidence interval 1.049-2.054, P = 0.025). CONCLUSION: An effective health policy for hematuria screening is needed for older age groups. A strategy of careful checkups and counseling regarding renal function is necessary for patients with isolated hematuria.

Sexually transmissible infections in middle and high school students: experience rates, risk factors and relationship with mental health - results from the Korean youth risk behaviour web-based survey.

UNLABELLED: Background Sexually transmissible infections (STIs) in adolescents are an important heath problem. However, the understanding of STIs among adolescents is poor. Rates of and risk factors for ever having a STI and relationships between ever having a STI and mental health in adolescents was investigated. METHODS: Data from the Korea Youth Risk Behaviour Web-based Survey (KYRBS) were used. The KYRBS is an anonymous, self-administered online survey. To achieve a representative sample of Koreans, researchers used a stratification, clustering and multi-stage sampling method. Data from adolescents who had not had sexual intercourse and surveys with missing data concerning sexual experience were excluded. Finally, 31363 participants were included. RESULTS: The rate of ever having a STI was 10.0%. A total of 26.5% of adolescents who have had sexual experience report always using contraceptives, and condoms are the most popular contraceptives (69.0%). Contraceptive method (condom, OR: 0.601, 95% CI: 0.491-0.736) and elementary school sexual debut (elementary school, OR: 1.000, middle school, OR: 0.235, 95% CI: 0.181-0.305; high school, OR: 0.128, 95% CI: 0.094-0.173) were significantly correlated with ever having a STI in the multivariate analysis. Depressed mood (OR: 1.379, 95% CI: 1.130-1.683), suicidal ideation (OR: 1.358, 95% CI: 1.109-1.664) and suicide attempts (OR: 1.382, 95% CI: 1.029-1.856) were significantly associated with ever having a STI after adjusting for potential confounders. CONCLUSIONS: STIs are common diseases in adolescents who have sexual experience and are significantly associated with mental health. Development of preventive measures and treatment policies including mental counselling for adolescents with STI are needed.