Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis.

A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately critical to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the molecular details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examination of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall molecular organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biological activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.

Recognition of protein-linked glycans as a determinant of peptidase activity.

The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

Extended Application of the Hybrid Procedure in Neonates with Left-Sided Obstructive Lesions in an Evolving Cardiac Program.

The hybrid approach to management of hypoplastic left heart syndrome (HLHS) was developed as an alternative to neonatal Norwood surgery, providing a less invasive initial palliation for HLHS. We describe our experience in extending the concept of the hybrid procedure to palliate neonates with anatomically compromised systemic arterial blood flow in a variety of congenital cardiac anomalies and supporting its application as first-line palliation in centers developing their HLHS programs. Retrospective review of patients undergoing therapy for HLHS at a single institution from June 2008 to December 2014 was performed. Subject demographics, clinical and procedural data, along with follow-up, were collected. Thirteen patients had initial hybrid palliation for HLHS during the time frame indicated at a median age of 8 days (range 1-29 days) and median weight of 3.4 kg (range 2.4-4.6 kg). Diagnoses included typical HLHS (n = 6), right-dominant unbalanced atrioventricular septal defect with arch hypoplasia (n = 4), double outlet right ventricle [subpulmonic VSD (n = 1) and intact ventricular septum (n = 1)] with hypoplastic transverse aortic arch and borderline left ventricular dimensions. Standard approach with bilateral pulmonary artery banding and ductal stenting was carried out in all thirteen patients. Two patients required two ductal stents at the time of index procedure. There were no intraprocedural complications. Median intubation length post-procedure was 4 days (range 1-74 days). Median hospital stay post-procedure was 47 days (range 15-270 days). The overall mortality rate on follow-up through comprehensive stage 2 over the 6-year experience was 38 % (5 out of 13). Of note, the mortality rate was significantly lower in the latter 3 years of the study period when the procedure was adopted as a primary palliation for HLHS (14 % or 1 out of 7) compared to the initial 3-year period when it was reserved for higher risk cohorts (67 % or 4 out of 6). Median time to subsequent surgery was 3 months (range 1-4 months). One patient required further ductal stenting on follow-up and developed subsequently airway compression. On median follow-up of 24 months, two patients required pulmonary artery arterioplasty. The hybrid procedure may be used for palliation for a variety of cardiac lesions to avoid high-risk surgery in the neonatal period. This approach may be also an alternative in centers performing lower number of Norwood surgery, which has been associated with higher mortality.

The cytotoxic and pro-apoptotic activities of the novel fluoropyrimidine F10 towards prostate cancer cells are enhanced by Zn(2+) -chelation and inhibiting the serine protease Omi/HtrA2.

BACKGROUND: Intracellular Zn(2+) levels decrease during prostate cancer progression and agents that modulate intracellular Zn(2+) are cytotoxic to prostate cancer cells by an incompletely described mechanism. F10 is a new polymeric fluoropyrimidine drug-candidate that displays strong activity with minimal systemic toxicity in pre-clinical models of prostate cancer and other malignancies. The effects of exogenous Zn(2+) or Zn(2+) chelation for enhancing F10 cytotoxicity are investigated as is the role of Omi/HtrA2, a serine protease that promotes apoptosis in response to cellular stress. METHODS: To test the hypothesis that the pro-apoptotic effects of F10 could be enhanced by modulating intracellular Zn(2+) we investigated cell-permeable and cell-impermeable Zn(2+) chelators and exogenous Zn(2+) and evaluated cell viability and apoptosis in cellular models of castration-resistant prostate cancer (CRPC; PC3, C4-2). The role of Omi/HtrA2 for modulating apoptosis was evaluated by pharmacological inhibition and Western blotting. RESULTS: Exogenous Zn(2+) initially reduced prostate cancer cell viability but these effects were transitory and were ineffective at enhancing F10 cytotoxicity. The cell-permeable Zn(2+) -chelator tetrakis-(2-pyridylmethl) ethylenediamine (TPEN) induced apoptosis in prostate cancer cells and enhanced the pro-apoptotic effects of F10. The pro-apoptotic effects of Zn(2+) -chelation in combination with F10 treatment were enhanced by inhibiting Omi/HtrA2 implicating this serine protease as a novel target for prostate cancer treatment. CONCLUSIONS: Zn(2+) -chelation enhances the pro-apoptotic effects of F10 and may be useful for enhancing the effectiveness of F10 for treatment of advanced prostate cancer. The serine protease Omi/HtrA2 modulates Zn(2+) -dependent apoptosis in prostate cancer cells and represents a new target for treatment of CRPC. Prostate 75:360-369, 2015. © 2014 Wiley Periodicals, Inc.

Site-specific DNA-doxorubicin conjugates display enhanced cytotoxicity to breast cancer cells.

Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-(15)N]-2'-deoxyguanosine in conjunction with [(1)H-(15)N] 2D NMR, while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life >30 h, considerably longer than alternative covalent and noncovalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anticancer drugs.

Structural analysis of CPF_2247, a novel α-amylase from Clostridium perfringens.

CPF_2247 from Clostridium perfringens ATCC 13124 was identified as a putative carbohydrate-active enzyme by its low sequence identity to endo-ß-1,4-glucanases belonging to family 8 of the glycoside hydrolase classification. The X-ray crystal structure of CPF_2247 determined to 2.0 Å resolution by single-wavelength anomalous dispersion using seleno-methionine-substituted protein revealed an (α/α)(6) barrel fold. A large cleft on the surface of the protein contains residues that are structurally conserved with key elements of the catalytic machinery in clan GH-M glycoside hydrolases. Assessment of CPF_2247 as a carbohydrate-active enzyme disclosed α-glucanase activity on amylose, glycogen, and malto-oligosaccharides.

Gaps in antihypertensive and statin treatments and benefits of optimisation: a modelling study in a 1 million ethnically diverse urban population in UK.

OBJECTIVES: To characterise gaps in antihypertensive treatment in people with hypertension and statin treatment in people with cardiovascular diseases (CVD) in a large urban population and quantify the health and economic impacts of their optimisation. DESIGN: A cross-sectional population study and a long-term CVD decision model. SETTING: Primary care, UK. PARTICIPANTS: All adults with diagnosed hypertension or CVD in a population of about 1 million people, served by 123 primary care practices in London, UK in 2019. INTERVENTIONS: Following UK clinical guidelines, all adults with diagnosed hypertension were categorised into optimal, suboptimal and untreated groups with respect to their antihypertensive treatment, and all adults with diagnosed CVD were categorised in the same manner with respect to their statin treatment. OUTCOMES: Proportion of patients suboptimally treated or untreated. Projected cardiovascular events avoided, years and quality-adjusted life years (QALYs) gained and healthcare costs saved with optimised treatments. RESULTS: 21 954 of the 91 828 adults with hypertension (24%; mean age 59 years; 49% women) and 9062 of the 23 723 adults with CVD (38%; mean age 69 years; 43% women) were not optimally treated with antihypertensive or statin treatment, respectively. Per 1000 additional patients optimised over 5 years, hypertension treatment is projected to prevent 25 (95% CI 16 to 32) major vascular events (MVEs) and 7 (3 to 10) vascular deaths, statin treatment, 28 (22 to 33) MVEs and 6 (4 to 7) vascular deaths. Over their lifespan, a patient with uncontrolled hypertension aged 60-69 years is projected to gain 0.64 (95% CI 0.36 to 0.87) QALYs with optimised hypertension treatment, and a similarly aged patient with previous CVD not optimally treated with statin is projected to gain 0.3 (0.24 to 0.37) QALYs with optimised statin treatment. In both cases, the hospital cost savings minus extra medication costs were about £1100 per person over remaining lifespan. CONCLUSIONS: Optimising cardiovascular treatments can cost-effectively reduce cardiovascular risk and improve life expectancy.

Opposing crosstalk between leptin and glucocorticoids rapidly modulates synaptic excitation via endocannabinoid release.

The hypothalamic paraventricular nucleus (PVN) integrates preautonomic and neuroendocrine control of energy homeostasis, fluid balance, and the stress response. We recently demonstrated that glucocorticoids act via a membrane receptor to rapidly cause endocannabinoid-mediated suppression of synaptic excitation in PVN neurosecretory neurons. Leptin, a major signal of nutritional state, suppresses CB(1) cannabinoid receptor-dependent hyperphagia (increased appetite) in fasting animals by reducing hypothalamic levels of endocannabinoids. Here we show that glucocorticoids stimulate endocannabinoid biosynthesis and release via a Galpha(s)-cAMP-protein kinase A-dependent mechanism and that leptin blocks glucocorticoid-induced endocannabinoid biosynthesis and suppression of excitation in the PVN via a phosphodiesterase-3B-mediated reduction in intracellular cAMP levels. We demonstrate this rapid hormonal interaction in both PVN magnocellular and parvocellular neurosecretory cells. Leptin blockade of the glucocorticoid-induced, endocannabinoid-mediated suppression of excitation was absent in leptin receptor-deficient obese Zucker rats. Our findings reveal a novel hormonal crosstalk that rapidly modulates synaptic excitation via endocannabinoid release in the hypothalamus and that provides a nutritional state-sensitive mechanism to integrate the neuroendocrine regulation of energy homeostasis, fluid balance, and the stress response.

Prostate-specific membrane antigen-targeted liposomes specifically deliver the Zn(2+) chelator TPEN inducing oxidative stress in prostate cancer cells.

AIM: To evaluate the potential use of zinc chelation for prostate cancer therapy using a new liposomal formulation of the zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). MATERIALS & METHODS: TPEN was encapsulated in nontargeted liposomes or liposomes displaying an aptamer to target prostate cancer cells overexpression prostate-specific membrane antigen. The prostate cancer selectivity and therapeutic efficacy of liposomal (targeted and nontargeted) and free TPEN were evaluated in vitro and in tumor-bearing mice. RESULTS & CONCLUSION: TPEN chelates zinc and results in reactive oxygen species imbalance leading to cell death. Delivery of TPEN using aptamer-targeted liposomes results in specific delivery to targeted cells. In vivo experiments show that TPEN-loaded, aptamer-targeted liposomes reduce tumor growth in a human prostate cancer xenograft model.

Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing.

Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10-6mol/l) relative to uncoated plates (2.4 × 10-6 mol/l), or plates coated with the related protein fibronectin (2.1 × 10-6 mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.

Thymineless death in F10-treated AML cells occurs via lipid raft depletion and Fas/FasL co-localization in the plasma membrane with activation of the extrinsic apoptotic pathway.

The polymeric fluoropyrimidine F10 displays excellent anti-leukemia activity in pre-clinical models of acute myelogenous leukemia (AML) through dual targeting of thymidylate synthase and DNA topoisomerase 1. Here we report that F10 activates the extrinsic apoptotic pathway in AML cells by enhancing localization of Fas and Fas ligand (FasL) at the plasma membrane and while reducing overall lipid raft levels promotes Fas/FasL co-localization in remaining lipid rafts. The HMG-CoA synthase inhibitor simvastatin was synergistic with F10 and induced cell death via similar apoptotic processes. Our results are consistent with diverse processes activating a common apoptotic pathway characterized by reduced overall levels of lipid rafts and Fas/FasL co-localization in the plasma membrane, including in remaining lipid rafts which may play a role in both cell-survival and cell death signaling.

Development and application of molecular biomarkers for characterizing Caribbean Yellow Band Disease in Orbicella faveolata.

Molecular stress responses associated with coral diseases represent an under-studied area of cnidarian transcriptome investigations. Caribbean Yellow Band Disease (CYBD) is considered a disease of Symbiodinium within the tissues of the coral host Orbicella faveolata. There is a paucity of diagnostic tools to assist in the early detection and characterization of coral diseases. The validity of a diagnostic test is determined by its ability to distinguish host organisms that have the disease from those that do not. The ability to detect and identify disease-affected tissue before visible signs of the disease are evident would then be a useful diagnostic tool for monitoring and managing disease outbreaks. Representational Difference Analysis (RDA) was utilized to isolate differentially expressed genes in O. faveolata exhibiting CYBD. Preliminary screening of RDA products identified a small number of genes of interest (GOI) which included an early growth response factor and ubiquitin ligase from the coral host as well as cytochrome oxidase from the algal symbiont. To further characterize the specificity of response, quantitative real-time PCR (qPCR) was utilized to compare the expression profiles of these GOIs within diseased tissues (visible lesions), tissues that precede visible lesions by 2-4 cm (transition area), and tissues from healthy-looking colonies with no signs of disease. Results show there are distinctive differences in the expression profiles of these three GOIs within each tissue examined. Collectively, this small suite of GOIs can provide a molecular "finger print" which is capable of differentiating between infected and uninfected colonies on reefs where CYBD is known to occur.

Dimeric DNA Aptamer Complexes for High-capacity-targeted Drug Delivery Using pH-sensitive Covalent Linkages.

Treatment with doxorubicin (Dox) results in serious systemic toxicities that limit effectiveness for cancer treatment and cause long-term health issues for cancer patients. We identified a new DNA aptamer to prostate-specific membrane antigen (PSMA) using fixed sequences to promote Dox binding and developed dimeric aptamer complexes (DACs) for specific delivery of Dox to PSMA(+) cancer cells. DACs are stable under physiological conditions and are internalized specifically into PSMA(+) C4-2 cells with minimal uptake into PSMA-null PC3 cells. Cellular internalization of DAC was demonstrated by confocal microscopy and flow cytometry. Covalent modification of DAC with Dox (DAC-D) resulted in a complex with stoichiometry ~4:1. Dox was covalently bound in DAC-D using a reversible linker that promotes covalent attachment of Dox to genomic DNA following cell internalization. Dox was released from the DAC-D under physiological conditions with a half-life of 8 hours, sufficient for in vivo targeting. DAC-D was used to selectively deliver Dox to C4-2 cells with endosomal release and nuclear localization of Dox. DAC-D was selectively cytotoxic to C4-2 cells with similar cytotoxicity as the molar equivalent of free-Dox. In contrast, DAC-D displayed minimal cytotoxicity to PC3 cells, demonstrating the complex displays a high degree of selectivity for PSMA(+) cells. DAC-D displays specificity and stability features that may be useful for improved delivery of Dox selectively to malignant tissue in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e107; doi:10.1038/mtna.2013.37; published online 16 July 2013.

Carbohydrate recognition by an architecturally complex α-N-acetylglucosaminidase from Clostridium perfringens.

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-D-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-ß-D-glucosamine-α-1,4-D-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-ß-D-glucosamine-α-1,4-D-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract.